Brachyury protein, non-poxvirus non-yeast vectors encoding Brachyury protein, and their use

ABSTRACT

Brachyury protein can be used to induce Brachyury-specific CD4+ T cells in vivo and ex vivo. It is also disclosed that Brachyury protein can be used to stimulate the production of both Brachyury-specific CD4+ T cells and Brachyury-specific CD8+ T cells in a subject, such as a subject with cancer. In some embodiments, the methods include the administration of a Brachyury protein. In additional embodiments, the methods include the administration of a nucleic acid encoding the Brachyury protein, such as in a non-pox non-yeast vector. In further embodiments, the method include the administration of host cells expressing the Brachyury protein.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application is a divisional application of U.S. patentapplication Ser. No. 14/428,308, filed Mar. 13, 2015, now abandoned,which claims the benefit of U.S. national phase of International PatentApplication No. PCT/US2013/059737, filed Sep. 13, 2013, which claims thebenefit of U.S. Provisional Patent Application No. 61/701,525, filedSep. 14, 2012, each of which is incorporated by reference in itsentirety herein.

SEQUENCE LISTING

Incorporated by reference in its entirety herein is a nucleotide/aminoacid sequence listing submitted concurrently herewith and identified asfollows: One 14,638 Byte ASCII (Text) file named “740352_ST25.TXT,”created on Aug. 21, 2018.

The nucleic and amino acid sequences listed in the accompanying sequencelisting are shown using standard letter abbreviations for nucleotidebases, and three letter code for amino acids, as defined in 37 C.F.R.1.822. Only one strand of each nucleic acid sequence is shown, but thecomplementary strand is understood as included by any reference to thedisplayed strand. In the accompanying sequence listing:

SEQ ID NO: 1 is an amino acid sequence of a human Brachyury protein.

SEQ ID NO: 2 is a nucleic acid sequence encoding a human Brachyuryprotein.

SEQ ID NO: 3 is an amino acid sequence of a murine Brachyury protein.

SEQ ID NO: 4 is a nucleic acid sequence encoding a murine Brachyuryprotein.

SEQ ID NO: 5 is a Brachyury class IIA epitope.

SEQ ID NO: 6 is a Brachyury class IIB epitope.

FIELD

This application relates to the field of cancer therapeutics,specifically to the use of a Brachyury protein and non-poxvirus,non-yeast vectors encoding a Brachyury protein for the treatment ofcancer.

BACKGROUND

The Brachyury gene was initially cloned from mouse developmental mutantscharacterized by an arrest in mesoderm formation (Hermann et al, Nature1990; 343:617-22) has been recognized as gene that is important inmesoderm development during gastrulation. Brachyury is a member of afamily of transcription factors, designated T-box transcription factors,these factors are characterized by a conserved DNA-binding domain(Papaioannou et al., Bioessays 1998; 20:9-19). These transcriptionfactors play an essential role in the formation and organization ofmesoderm in vertebrates (see, for example, Edwards et al., Genome Res1996; 6:226-33). In addition to the important role of the T-box proteinsin the control of developmental processes, several members of thisfamily are deregulated in cancer. For example, the human Tbx2 gene hasbeen reported to be amplified in pancreatic cancer cell lines (Mahlamakiet al., Genes Chromosomes Cancer 2002, 35:353-8) and is overexpressed inBRCA-1- and BRCA-2-mutated breast tumors (Sinclair et al., Cancer Res2002; 62:3587-91). In addition, Tbx3 expression has been shown to beaugmented in certain human breast cancer cell lines (Fan et al., CancerRes 2004; 64:5132-9). Expression of Brachyury has also been documentedin human teratocarcinoma lines: a subset of germ cell tumors,teratocarcinomas are embryonal carcinoma cells with competence formesoderm differentiation (Gokhale et al., Cell Growth Differ 2000;11:157-62) and in chordomas (see, for example, Vojovic et al., J Pathol2006; 209:157-65).

Immunotherapeutic interventions against cancer depend on theidentification of tumor antigens able to elicit a host immune responseagainst the tumor cells. Good targets are molecules that are selectivelyexpressed by malignant cells and that are also essential for malignanttransformation and/or tumor progression. The epithelial-mesenchymaltransition (EMT) has been recognized as a key step during theprogression of primary tumors into metastases (Thiery et al., Nat RevCancer 2002; 2:442-54). Several molecules have been identified that playa key role in EMT during tumor progression (Huber et al., Curr Opin CellBiol 2005; 17:548-58), among them the transcription factors Twist,Snail, and Slug (Yang et al., Cell 2004; 117:927-39; Cano et al., NatCell Biol 2000; 2: 76-83). Molecules that trigger EMT could function toprevent tumor invasion and metastasis. However, a need remains forreagents that induce an effective immune response to cancer, including aCD4 and a CD8 T cell response.

SUMMARY

It is disclosed herein that Brachyury protein or a Brachyury polypeptidecan be used to induce Brachyury-specific CD4+ T cells in vivo and exvivo. It is also disclosed that Brachyury protein and Brachyurypolypeptides can be used to stimulate the production of bothBrachyury-specific CD4+ T cells and Brachyury-specific CD8+ T cells.Brachyury is expressed in numerous human cancers, such as in cancer ofthe small intestine, stomach, kidney bladder, uterus, ovary, testes,lung, colon, prostate, bronchial tube, chronic lymphocytic leukemia(CLL), other B cell-based malignancies, and breast cancer, such asinfiltrating ductal carcinomas of the breast. Thus, Brachyury protein,Brachyury polypeptides, and nucleic acids encoding Brachyury proteinand/or polypeptides, can be used to produce Brachyury specific CD4+ Tcells, and CD8+ T cells, that can be used for the treatment orprevention of cancer.

In some embodiments, methods are disclosed for inducing CD4+Brachyury-specific T cells and/or CD8+ Brachyury specific T cells. Themethods include the use of a Brachyury protein, a Brachyury polypeptide,nucleic acids encoding the Brachyury protein and/or Brachyurypolypeptides, or host cells expressing the Brachyury protein orpolypeptide, such as such as a Salmonella or Listeria host cells. Theseagents can be administered either alone or in conjunction with anotheragent, such as a cytokine and/or another cancer therapy. In someembodiments, methods are disclosed for treating a subject with a cancer,such as a breast cancer, cancer of the small intestine, stomach, kidney,bladder, uterus, ovaries, testes lung, colon or prostate, or a tumor ofB cell origin, or for preventing these cancers in a subject. In someembodiments, the methods include measuring Brachyury-specific CD4+ Tcells. In further embodiments, the methods also induce CD8+ Brachyuryspecific T cells.

Non-pox non-yeast vectors encoding a Brachyury protein are disclosedthat can be used to induce CD4+ Brachyury-specific T cells and/or CD8+Brachyury-specific T cells. In some non-limiting examples, the vector isan alphavirus, a lentivirus, an adenovirus, a measles virus or apoliovirus vector. In additional embodiments, host cells transformedwith these vectors, such as Salmonella and Listeria host cells areprovided.

In additional embodiments, methods are provided for inhibiting thegrowth of a cancer cell in a subject. These methods include contacting adendritic cell with a protein comprising an amino acid sequence at least90% identical to the amino acid sequence set forth as SEQ ID NO: 1, apolypeptide comprising at least 15 consecutive amino acids of the aminoacid sequence set forth at SEQ ID NO: 1 that specifically binds a MajorHistocompatibility Class (MHC class II) molecule, or a Listeria orSalmonella host cell expressing the protein or the polypeptide therebypreparing a specific antigen presenting cell. These methods also includeadministering the antigen presenting cell to the subject, therebyinducing an immune response and inhibiting the growth of the cancercell.

The foregoing and other features and advantages will become moreapparent from the following detailed description of several embodiments,which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Brachyury-specific CD4+ T cells can be expanded from PBMCs ofnormal donors by culture in the presence of purified recombinantBrachyury protein. Dendritic cells (DCs) from 2 normal donors wereprepared by culture in the presence of GM-CSF and IL-4. On day 5, apurified recombinant Brachyury protein was added (10 g/ml) for 48 hours.For donor 2, an additional culture was set up using purified HSA (humanserum albumin) control protein (10 μg/ml). On day 7, protein-pulsed DCswere harvested, irradiated (20 Gy) and used as antigen-presenting cells(APCs) to stimulate autologous PBMCs (ratio DC:PBMCs equal to 1:10). Ondays 3 and 5, IL-2 (20 U/ml) was added to the cultures. T cells wereharvested on day 7 and CD4+ T cells were isolated by negative selectionwith magnetic beads. CD4+ T cells were stimulated in similar manner foran additional 7-day cycle. On day 7, CD4+ T cells were re-isolated byusing magnetic beads and evaluated for IFN-gamma production in responseto autologous, irradiated PBMCs (ratio PBMCs:T cells equal to 3:1) aloneor pulsed with control HSA protein vs. Brachyury protein (10 μg/ml).Culture supernatants were collected at 96 hours and evaluated forIFN-gamma by ELISA.

FIG. 2. A Brachyury-specific CD4 T cell line releases cytokines andchemokines when stimulated with a class-II DRB1*0401 binding Brachyurypeptide. Brachyury class IIA epitope (SEQ ID NO: 5) and Brachyury classIIB epitope (SEQ ID NO: 6).

FIG. 3A-3E. Brachyury induces an epithelial-to-mesenchymal transition(EMT) in breast carcinoma cells. (A) MCF7-pcDNA and MCF7-phBrachyurystable transfectants grown on plastic surface for bright field images(top panels) and grown immunofluorescence analysis of E-cadherinexpression (green signal); blue signal represents DAPI-stained nuclei(bottom panels). (B) Membrane images from in vitro cell migration (toppanels) and ECM invasion assays (bottom panels) for MCF7-pcDNA andMCF7-pBrachyury cells. Results are representative of three experiments.(C, D) Real-time PCR was performed on indicated cell pairs forBrachyury, Fibronectin, and Vimentin. Values (mean±SEM) are expressed asa ratio to the endogenous control GAPDH. (E) Immunofluorescent analysisof Fibronectin expression in MDA-MB-436-con.shRNA andMDA-MB-436-Br.shRNA stable transfectants (original magnification 20×).The green signal represents staining for Fibronectin; the blue signalrepresents the DAPI-stained nuclei.

FIGS. 4A-4D. Effect of Brachyury expression on stem cell markerexpression and mammosphere growth of tumor cells. Real-time PCR wasperformed for indicated genes on cDNA from (A) MCF7-pcDNA andMCF7-phBrachyury cells and (C) MDA-MB-436-con.shRNA andMDA-MB-436-Br.shRNA cells. Values (mean±SEM) are expressed as a ratio tothe endogenous control GAPDH. Mammospheres were grown from the MCF7 (B)or the MDA-MB-436 (D) tumor cell pairs on ultra-low-attachment plates.Primary mammospheres were dissociated and re-plated for secondarycultures. Bright field images of mammospheres at 10× magnification andmean number of mammospheres per 10× microscope field are shown forsecondary cultures in the left and right panels, respectively. Errorbars indicate SEM of 8-10 measurements.

FIGS. 5A-5D. Expression of Brachyury mRNA in breast carcinoma tissues.(A) Real-time PCR was performed for Brachyury, Twist, Snail, and Slug onhuman breast primary tumor tissue cDNA from 41 breast cancer patients.As controls, 7 samples of normal breast cDNA were also analyzed, eachobtained from a histologically normal section of breast from a patientwith cancer or fibrocystic disease. (B) Real-time PCR was performed forBrachyury on human primary breast tumor tissue cDNA from 107 invasiveductal adenocarcinomas, 6 invasive lobular adenocarcinomas, and 5 mixedductal/lobular adenocarcinomas. As controls, 7 samples of normal breastcDNA were also analyzed, each obtained from a histologically normalsection of breast from a patient with cancer or fibrocystic disease. Allvalues and the means for each group are expressed as a ratio to theendogenous control GAPDH. Brachyury expression is shown for (B) breastprimary tumor tissues from stages I-III grouped together, (C) breastprimary tumor tissues grouped by histological tumor grade (Nottinghamgrading), (D) breast primary tumor tissues grouped by ER and PRexpression (ER+PR+ versus ER−PR−).

FIGS. 6A-6F. Immunohistochemical detection of Brachyury in primarybreast carcinoma and metastatic tissues. Transmitted lightphotomicrographs of tissue sections stained for Brachyury expression in(A) a primary infiltrating ductal carcinoma, Grade 3 (patient 11); (B) aprimary infiltrating ductal carcinoma, Grade 3 and (C) correspondinglymph node metastasis from the same patient (patient 6); (D, E) bonemetastatic lesions from two different breast cancer patients (patients22 and 23); (F) brain metastatic lesion from a breast cancer patient(patient 24). The brown signal represents staining for Brachyury.Magnification 20× (A-F).

FIG. 7A-7C. Immunogenicity of Brachyury. (A) Detection of IgG antibodiesagainst Brachyury in the serum of normal donors and metastatic breastcancer patients. Shown is the number of positive cases in each group,stratified by titer of IgG as determined by ELISA assay. Statisticalanalysis was performed, comparing breast vs. normal donors.Brachyury-specific CTLs were generated from the peripheral blood of aprostate cancer patient via stimulation with a Brachyury-derivedpeptide. Cytotoxic activity was assessed in a 16-h assay against (B)HLA-A2⁺/Brachyury⁺ MCF7 cells or HLA-A2⁻/Brachyury⁺ MDA-MB-436 cells,and (C) HLA-A2⁺/Brachyury⁺ MDA-MB-231 cells. The effector-to-target(E:T) ratios are indicated; major histocompatibility complex(MHC)—restriction was analyzed by pre-incubation of the targets withcontrol IgG or a HLA-A specific antibody.

DETAILED DESCRIPTION

It is disclosed herein that Brachyury protein and Brachyury polypeptidesof greater than 15 amino acids in length can be used to induceBrachyury-specific CD4+ T cells in vivo and ex vivo. It is alsodisclosed that Brachyury protein and Brachyury polypeptides can be usedto stimulate the production of both Brachyury-specific CD4+ T cells andBrachyury-specific CD8+ T cells. Brachyury protein is expressed innumerous human cancers, such as cancer of the small intestine, stomach,kidney bladder, uterus, ovary, testes, lung, colon, prostate, bronchialtube, chronic lymphocytic leukemia (CLL), other B cell-basedmalignancies and breast cancer, such as infiltrating ductal carcinomasof the breast and thus the method disclosed herein can be used to treator prevent these cancers. In specific non-limiting examples, the breastcancer is an estrogen receptor negative and progesterone receptornegative breast cancer. In additional non-limiting examples, the canceris any cancer that is radiation resistant and/or chemotherapy resistant.The cancer can express Brachyury or have the potential to expressBrachyury.

Non-pox non-yeast vectors encoding a Brachyury protein or a Brachyurypolypeptide, and host cells expressing Brachyury are disclosed, thesevectors and host cells can be used to induce CD4+ Brachyury-specific Tcells and/or CD8+ T cells. In some non-limiting examples, these vectorsare adenovirus vectors, alphavirus vectors, lentivirus vectors,poliovirus vectors, Listeria vectors, Salmonella vectors or measlesvirus vectors. In additional embodiments, host cells transformed withthese vectors, and methods of using these proteins, polynucleotides,vectors, and host cells are provided. In some examples the host cellsare Salmonella or Listeria host cells.

Thus, methods are provided for inducing CD4+ Brachyury-specific T cellsand/or CD8+ T cells. The methods include the use of a Brachyury protein,Brachyury polypeptide, dendritic cells expressing Brachyury epitopes,nucleic acids encoding Brachyury protein and/or polypeptides, includingnon-pox non-yeast vectors encoding the Brachyury protein and/or theBrachyury polypeptide to induce the production of CD4+ Brachyuryspecific T cells. In some embodiments, methods are disclosed fortreating a subject having cancer, such as, but not limited to, a cancerof the small intestine, stomach, kidney bladder, uterus, ovary, testes,lung, colon, prostate, bronchial tube, chronic lymphocytic leukemia(CLL), other B cell-based malignancies, or breast cancer, such as aninfiltrating ductal carcinoma or estrogen receptor negative andprogesterone receptor negative breast cancers. Any of these cancers canbe chemotherapy resistant and/or radiation resistant. The cancer canexpress Brachyury or have the potential to express Brachyury. Methodsare also disclosed for preventing these cancers.

These methods include inducing CD4+ Brachyury-specific T cells; themethod can also include inducing CD8+ Brachyury-specific T cells. TheBrachyury protein, Brachyury polypeptide, dendritic cells, nucleic acid,or non-pox non-yeast vector encoding the Brachyury protein can beadministered to the subject either alone or in conjunction with a secondagent, such as radiation therapy and/or chemotherapy.

In some embodiments, the Brachyury protein comprises an amino acidsequence at least 90% identical, or at least 95% identical, to the aminoacid sequence set forth as SEQ ID NO: 1. In other embodiments, theBrachyury protein comprises, or consists of, the amino acid sequence setforth as SEQ ID NO: 1, the amino acid sequence set forth as SEQ ID NO: 1without the N-terminal methionine, or the amino acid sequence set forthas SEQ ID NO: 1, with substitutions at position 177 (Asp vs. Gly,respectively), position 368 (Thr vs. Ser, respectively) and position 409(Asn vs. Asp, respectively).

In further embodiments, a Brachyury polypeptide comprises at least 15amino acids of the amino acid sequence set forth as SEQ ID NO: 1, suchas at least 20, at least 30, at least 40, at least 50, at least 60, atleast 70, at least 80, at least 90, at least 100, at least 200 aminoacids of the amino acid sequence set forth as SEQ ID NO: 1, wherein theentirety of SEQ ID NO: 1 is not included in the polypeptide. Inadditional embodiments, a Brachyury polypeptide is 15 to 100 amino acidsof SEQ ID NO: 1, such as 15 to 200 amino acids, 15 to 300 amino acids,15 to 400 amino acids, or 15 to 435 amino acids of SEQ ID NO: 1.

In additional embodiments, methods are provided for inhibiting thegrowth of a cancer cell in a subject. These methods include contacting adendritic cell with a protein comprising an amino acid sequence at least90% identical to the amino acid sequence set forth as SEQ ID NO: 1, apolypeptide comprising at least 15 consecutive amino acids of the aminoacid sequence set forth at SEQ ID NO: 1 that specifically binds a MajorHistocompatibility Class (MHC class II) molecule, or a Listeria orSalmonella host cell expressing the protein, thereby preparing aspecific antigen presenting cell. These methods also includeadministering the antigen presenting cell to the subject, therebyinducing an immune response and inhibiting the growth of the cancercell.

Terms

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology maybe found in Benjamin Lewin, Genes V, published by Oxford UniversityPress, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), TheEncyclopedia of Molecular Biology, published by Blackwell Science Ltd.,1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biologyand Biotechnology: a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8).

In order to facilitate review of the various embodiments of thisdisclosure, the following explanations of specific terms are provided:

Adenovirus: A virus of the family Adenoviridae, which are medium-sized(90-100 nm), nonenveloped icosahedral viruses composed of a nucleocapsidand a double-stranded linear DNA genome. The adenovirus genome islinear, non-segmented double-stranded (ds) DNA that is between 26 and 45kb. This allows the virus to theoretically carry 22 to 40 genes. Thelinear dsDNA genome is able to replicate in the nucleus of mammaliancells using the host's replication machinery. However, adenoviral DNAdoes not integrate into the genome and is not replicated during celldivision.

Adeno-associated Virus: Adeno-associated virus (AAV) is a small virusthat infects humans and some other primate species. AAV is not currentlyknown to cause disease and consequently the virus causes a very mildimmune response. AAV can infect both dividing and non-dividing cells andmay incorporate its genome into that of the host cell. The AAV genome isbuilt of single-stranded deoxyribonucleic acid (ssDNA), either positive-or negative-sensed, which is about 4.7 kilobase long. The genomecomprises inverted terminal repeats (ITRs) at both ends of the DNAstrand, and two open reading frames (ORFs): rep and cap. Rep is composedof four overlapping genes encoding Rep proteins required for the AAVlife cycle, and Cap contains overlapping nucleotide sequences of capsidproteins: VP1, VP2 and VP3, which interact together to form a capsid ofan icosahedral symmetry. For gene therapy, ITRs seem to be the onlysequences required in cis next to the therapeutic gene: structural (cap)and packaging (rep) genes can be delivered in trans.

Adjuvant: A vehicle used to enhance antigenicity. Adjuvants include asuspension of minerals (alum, aluminum hydroxide, or phosphate) on whichantigen is adsorbed; or water-in-oil emulsion in which antigen solutionis emulsified in mineral oil (Freund incomplete adjuvant), sometimeswith the inclusion of killed mycobacteria (Freund's complete adjuvant)to further enhance antigenicity (inhibits degradation of antigen and/orcauses influx of macrophages). Immunostimulatory oligonucleotides (suchas those including a CpG motif) can also be used as adjuvants (forexample see U.S. Pat. Nos. 6,194,388; 6,207,646; 6,214,806; 6,218,371;6,239,116; 6,339,068; 6,406,705; and 6,429,199). Adjuvants includebiological molecules (a “biological adjuvant”), such as costimulatorymolecules. Exemplary adjuvants include IL-2, RANTES, GM-CSF, TNF-α,IFN-γ, G-CSF, LFA-3, CD72, B7-1, B7-2, OX-40L and 4-1 BBL. Anotherexemplary adjuvant is chitosan. Another adjuvant isBacillus-Calmette-Guerin adjuvant.

Alphavirus: A virus that belongs to the group IV Togaviridae family ofviruses. The alphaviruses are small, spherical, enveloped viruses with agenome of a single positive sense strand RNA. The total genome lengthranges between 11,000 and 12,000 nucleotides, and has a 5′ cap, and 3′poly-A tail. The four non-structural protein genes are encoded in the 5′two-thirds of the genome, while the three structural proteins aretranslated from a subgenomic mRNA colinear with the 3′ one-third of thegenome. The alphaviruses include the Ross River virus, Sindbis virus,Semliki Forest virus, and Venezuelan equine encephalitis virus.

Antigen: A compound, composition, or substance that can stimulate theproduction of antibodies or a T cell response in an animal, includingcompositions that are injected or absorbed into an animal. An antigenreacts with the products of specific humoral or cellular immunity,including those induced by heterologous immunogens. The term “antigen”includes all related antigenic epitopes. “Epitope” or “antigenicdeterminant” refers to a site on an antigen to which B and/or T cellsrespond. In one embodiment, T cells respond to the epitope, when theepitope is presented in conjunction with an MHC molecule. Epitopes canbe formed both from contiguous amino acids or noncontiguous amino acidsjuxtaposed by tertiary folding of a protein. Epitopes formed fromcontiguous amino acids are typically retained on exposure to denaturingsolvents whereas epitopes formed by tertiary folding are typically loston treatment with denaturing solvents. An epitope typically includes atleast 3, and more usually, at least 5, about 9, or about 8-10 aminoacids in a unique spatial conformation, but is generally not more than20 amino acids in length. Methods of determining spatial conformation ofepitopes include, for example, x-ray crystallography and 2-dimensionalnuclear magnetic resonance.

An antigen can be a tissue-specific antigen, or a disease-specificantigen. These terms are not exclusive, as a tissue-specific antigen canalso be a disease specific antigen. A tissue-specific antigen isexpressed in a limited number of tissues, such as a single tissue.Specific, non-limiting examples of a tissue specific antigen are aprostate specific antigen, a uterine specific antigen, and/or a testesspecific antigen. A tissue specific antigen may be expressed by morethan one tissue, such as, but not limited to, an antigen that isexpressed in more than one reproductive tissue, such as in both prostateand uterine tissue. A disease-specific antigen is expressedcoincidentally with a disease process. Specific non-limiting examples ofa disease-specific antigen are an antigen whose expression correlateswith, or is predictive of, tumor formation, such as prostate cancerand/or uterine cancer and/or testicular cancer. A disease-specificantigen can be an antigen recognized by T cells or B cells.

Amplification: Of a nucleic acid molecule (e.g., a DNA or RNA molecule)refers to use of a technique that increases the number of copies of anucleic acid molecule in a specimen. An example of amplification is thepolymerase chain reaction, in which a biological sample collected from asubject is contacted with a pair of oligonucleotide primers, underconditions that allow for the hybridization of the primers to a nucleicacid template in the sample. The primers are extended under suitableconditions, dissociated from the template, and then re-annealed,extended, and dissociated to amplify the number of copies of the nucleicacid. The product of amplification can be characterized byelectrophoresis, restriction endonuclease cleavage patterns,oligonucleotide hybridization or ligation, and/or nucleic acidsequencing using standard techniques. Other examples of amplificationinclude strand displacement amplification, as disclosed in U.S. Pat. No.5,744,311; transcription-free isothermal amplification, as disclosed inU.S. Pat. No. 6,033,881; repair chain reaction amplification, asdisclosed in WO 90/01069; ligase chain reaction amplification, asdisclosed in EP-A-320 308; gap filling ligase chain reactionamplification, as disclosed in U.S. Pat. No. 5,427,930; and NASBA™ RNAtranscription-free amplification, as disclosed in U.S. Pat. No.6,025,134.

Antibody: Immunoglobulin molecules and immunologically active portionsof immunoglobulin molecules, i.e., molecules that contain an antigenbinding site that specifically binds (immunoreacts with) an antigen,such as Brachyury protein.

A naturally occurring antibody (e.g., IgG, IgM, IgD) includes fourpolypeptide chains, two heavy (H) chains and two light (L) chainsinterconnected by disulfide bonds. However, it has been shown that theantigen-binding function of an antibody can be performed by fragments ofa naturally occurring antibody. Thus, these antigen-binding fragmentsare also intended to be designated by the term “antibody.” Specific,non-limiting examples of binding fragments encompassed within the termantibody include (i) a Fab fragment consisting of the V_(L), V_(H),C_(L) and C_(H1) domains; (ii) an F_(d) fragment consisting of the V_(H)and C_(H1) domains; (iii) an Fv fragment consisting of the VL and VHdomains of a single arm of an antibody, (iv) a dAb fragment (Ward etal., Nature 341:544-546, 1989) which consists of a V_(H) domain; (v) anisolated complementarity determining region (CDR); and (vi) a F(ab′)₂fragment, a bivalent fragment comprising two Fab fragments linked by adisulfide bridge at the hinge region.

Immunoglobulins and certain variants thereof are known and many havebeen prepared in recombinant cell culture (e.g., see U.S. Pat. Nos.4,745,055; 4,444,487; WO 88/03565; EP 256,654; EP 120,694; EP 125,023;Faoulkner et al., Nature 298:286, 1982; Morrison, J. Immunol. 123:793,1979; Morrison et al., Ann Rev. Immunol 2:239, 1984). Humanizedantibodies and fully human antibodies are also known in the art.

Animal: Living multi-cellular vertebrate organisms, a category thatincludes, for example, mammals and birds. The term mammal includes bothhuman and non-human mammals. Similarly, the term “subject” includes bothhuman and veterinary subjects.

Brachyury: The Brachyury gene is known to be important for thedevelopment of mesoderm during gastrulation. Brachyury is the foundingmember of a family of transcription factors, designated T-boxtranscription factors, characterized by a conserved DNA-binding domain(Papaioannou and Silver, Bioessays 20(1):9-19, 1998), that has anessential role in the formation and organization of mesoderm invertebrates (see, for example, Kispert and Herrmann, Embo J12(8):3211-20, 1993). For example, in Xenopus, Brachyury is anearly-immediate response gene of mesoderm inducers, such as activin orTGF-β, and injection of Brachyury mRNA in embryos is sufficient toinduce ectopic mesoderm development (Smith et al., Cell 67(1):79-87,1991). In addition to the fundamental role of the T-box proteins in thecontrol of developmental processes, several members of this familyappear to be deregulated in cancer. The human Tbx2 gene has beenreported to be amplified in pancreatic cancer cell lines (Mahlamaki etal., Genes Chromosomes Cancer 35(4):353-8, 2002) and over-expressed inBRCA-1- and BRCA-2-mutated breast tumors (Sinclair et al., Cancer Res62(13):3587-9, 2002). Brachyury expression has been reported in humanteratocarcinoma lines and chordomas (Vujovic et al, J Pathol 209(2):157-65, 2006). Exemplary human brachyury amino acid and nucleic acidsequences are set forth in GENBANK® Accession No NP_003172 and GENBANK®Accession No. NM_003181, as available on Feb. 23, 2007, incorporatedherein by reference, and are provided below.

Breast cancer: A neoplastic condition of breast tissue that can bebenign or malignant. The most common type of breast cancer is ductalcarcinoma. Ductal carcinoma in situ is a non-invasive neoplasticcondition of the ducts. Lobular carcinoma is not an invasive disease butis an indicator that a carcinoma may develop. Infiltrating (malignant)carcinoma of the breast can be divided into stages (I IIA, IIB, IIIA,IIIB, and IV). Tumor size staging and node involvement staging can becombined into a single clinical staging number, as exemplified below.

Tumor size staging Node involvement staging Clinical stage T1 N0 I T1 N1IIA T2 N0 IIA T2 N1 IIB T3 N0 IIB T1-T2 N2 IIIA T3 N1 IIIA T3 N2 IIIA T4N0-N2 IIIB

Breast carcinomas lose the typical histology and architecture of normalbreast glands. Generally, carcinoma cells overgrow the normal cells andlose their ability to differentiate into glandular like structures. Thedegree of loss of differentiation in general is related to theaggressiveness of the tumor. For example, “in situ” carcinoma bydefinition retains the basement membrane intact, whereas as itprogresses to “invasive”, the tumor shows breakout of basementmembranes. Thus one would not expect to see, within breast carcinomas,staining of a discrete layer of basal cells as seen in normal breasttissue. For a discussion of the physiology and histology of normalbreast and breast carcinoma, see Ronnov-Jessen, L., Petersen, O. W. &Bissell, M. J. Cellular changes involved in conversion of normal tomalignant breast: importance of the stromal reaction. Physiol Rev 76,69-125 (1996).

Breast cancers can be divided into groups based on their expressionprofiles. Basal-type carcinomas usually are negative for expression ofestrogen receptor (ER) and negative for expression of HER2 (erbB2) andprogesterone receptor (PR), and thus are referred to as “triple-negativebreast cancers” or “TNBC.” This type of breast cancer is also denotedER⁻/HER2⁻/PR⁻ and represents about 15-20% of all breast cancer, andgenerally cannot be treated using Her2 targeted or estrogen targetedtherapies. It is believed that the aggressive nature of this cancer iscorrelated with an enrichment for cancer stem cells (CSC) with aCD44⁺CD24^(−/lo) phenotype. In some embodiments, basal carcinomas arenegative for expression of progesterone receptor (PR), positive forexpression of epidermal growth factor receptor (EGFR), and positive forexpression of cytokeratin 5 (CK5). This phenotype is denoted as follows:ER⁻/PR⁻/HER2⁻/CK5⁺/EGFR⁺.

Cancer or Tumor: A malignant neoplasm that has undergone characteristicanaplasia with loss of differentiation, increased rate of growth,invasion of surrounding tissue, and is capable of metastasis. Forexample, prostate cancer is a malignant neoplasm that arises in or fromprostate tissue, ovarian cancer is a malignant neoplasm that arises inor from ovarian tissue, colon cancer is a malignant neoplasm that arisesin or from colon tissue, and lung cancer is a malignant neoplasm thatarises in the lungs. Residual cancer is cancer that remains in a subjectafter any form of treatment given to the subject to reduce or eradicatethe cancer. Metastatic cancer is a cancer at one or more sites in thebody other than the site of origin of the original (primary) cancer fromwhich the metastatic cancer is derived. Cancer includes, but is notlimited to, sarcomas and carcinomas. Prostate cancer is a malignanttumor, generally of glandular origin, of the prostate. Prostate cancersinclude adenocarcinomas and small cell carcinomas.

cDNA (complementary DNA): A piece of DNA lacking internal, non-codingsegments (introns) and regulatory sequences that determinetranscription. cDNA is synthesized in the laboratory by reversetranscription from messenger RNA extracted from cells.

Chemotherapeutic agents: Any chemical agent with therapeutic usefulnessin the treatment of diseases characterized by abnormal cell growth. Suchdiseases include tumors, neoplasms, and cancer as well as diseasescharacterized by hyperplastic growth such as psoriasis. In oneembodiment, a chemotherapeutic agent is an agent of use in treatingbreast and/or prostate cancer. In one embodiment, a chemotherapeuticagent is radioactive compound. One of skill in the art can readilyidentify a chemotherapeutic agent of use (e.g. see Slapak and Kufe,Principles of Cancer Therapy, Chapter 86 in Harrison's Principles ofInternal Medicine, 14th edition, Perry et al., Chemotherapy, Ch. 17 inAbeloff, Clinical Oncology 2^(nd) ed., © 2000 Churchill Livingstone,Inc; Baltzer L, Berkery R (eds): Oncology Pocket Guide to Chemotherapy,2nd ed. St. Louis, Mosby-Year Book, 1995; Fischer D S, Knobf M F,Durivage H J (eds): The Cancer Chemotherapy Handbook, 4th ed. St. Louis,Mosby-Year Book, 1993). Combination chemotherapy is the administrationof more than one agent to treat cancer, such as the administration of anon-pox non-yeast vector encoding Brachyury in combination with aradioactive or chemical compound to a subject.

Conservative variants: “Conservative” amino acid substitutions are thosesubstitutions that do not substantially affect or decrease an activityor antigenicity of an antigenic epitope of Brachyury. Specific,non-limiting examples of a conservative substitution include thefollowing examples:

Original Residue Conservative Substitutions Al Ser Arg Lys Asn Gln, HisAsp Glu Cys Ser Gln Asn Glu Asp His Asn; Gln Ile Leu, Val Leu Ile; ValLys Arg; Gln; Glu Met Leu; Ile Phe Met; Leu; Tyr Ser Thr Thr Ser Trp TyrTyr Trp; Phe Val Ile; Leu

The term conservative variant also includes the use of a substitutedamino acid in place of an unsubstituted parent amino acid, provided thatantibodies raised to the substituted polypeptide also immunoreact withthe unsubstituted polypeptide, and/or that the substituted polypeptideretains the function of the unsubstituted polypeptide. Non-conservativesubstitutions are those that reduce an activity or antigenicity.

CD4: Cluster of differentiation factor 4, a T cell surface protein thatmediates interaction with the MHC Class II molecule. CD4 also serves asthe primary receptor site for HIV on T cells during HIV infection. Cellsthat express CD4 are often helper T cells.

CD8: Cluster of differentiation factor 8, a T cell surface protein thatmediates interaction with the MHC Class I molecule. Cells that expressCD8 are often cytotoxic T cells.

Consists Essentially Of/Consists Of: With regard to a polypeptide orprotein, a polypeptide (or protein) that consists essentially of aspecified amino acid sequence if it does not include any additionalamino acid residues. However, the polypeptide (or protein) can includeadditional non-peptide components, such as labels (for example,fluorescent, radioactive, or solid particle labels), sugars or lipids.With regard to a polypeptide or protein, a polypeptide or protein thatconsists of a specified amino acid sequence does not include anyadditional amino acid residues, nor does it include additionalnon-peptide components, such as lipids, sugars or labels.

Costimulatory molecule: Although engagement of the TCR with peptide-MHCdelivers one signal to the T cell, this signal alone can be insufficientto activate the T cell. Costimulatory molecules are molecules that, whenbound to their ligand, deliver a second signal required for the T cellto become activated. The most well-known costimulatory molecule on the Tcell is CD28, which binds to either B7-1 (also called CD80) or B7-2(also known as CD86). An additional costimulatory molecule is B7-3.Accessory molecules that also provide a second signal for the activationof T cells include intracellular adhesion molecule (ICAM-1 and ICAM-2),leukocyte function associated antigen (LFA-1, LFA-2 and LFA-3).Integrins and tumor necrosis factor (TNF) superfamily members can alsoserve as co-stimulatory molecules.

Degenerate variant: A polynucleotide encoding an epitope of Brachyurythat includes a sequence that is degenerate as a result of the geneticcode. There are 20 natural amino acids, most of which are specified bymore than one codon. Therefore, all degenerate nucleotide sequences areincluded in this disclosure as long as the amino acid sequence of theBrachyury protein encoded by the nucleotide sequence is unchanged.

Dendritic cell (DC): Dendritic cells are the principle antigenpresenting cells (APCs) involved in primary immune responses. Dendriticcells include plasmacytoid dendritic cells and myeloid dendritic cells.Their major function is to obtain antigen in tissues, migrate tolymphoid organs and present the antigen in order to activate T cells.Immature dendritic cells originate in the bone marrow and reside in theperiphery as immature cells.

Diagnostic: Identifying the presence or nature of a pathologiccondition, such as, but not limited to, a cancer, such as smallintestine, stomach, kidney, bladder, uterus, ovary, testes, lung, colonor prostate cancer. Diagnostic methods differ in their sensitivity andspecificity. The “sensitivity” of a diagnostic assay is the percentageof diseased individuals who test positive (percent of true positives).The “specificity” of a diagnostic assay is 1 minus the false positiverate, where the false positive rate is defined as the proportion ofthose without the disease who test positive. While a particulardiagnostic method may not provide a definitive diagnosis of a condition,it suffices if the method provides a positive indication that aids indiagnosis. “Prognostic” means predicting the probability of development(for example, severity) of a pathologic condition, such as prostatecancer, or metastasis.

Epithelial-to-Mesenchymal Transition: The epithelium is the covering ofinternal and external surfaces of the body, including the lining ofvessels and other small cavities, that consists of cells joined bybiological cementing substances. Generally, fully differentiatedepithelial cells express proteins characteristic of a differentiatedphenotype, such as insulin, and have a limited capacity to proliferate.The mesenchyme is the meshwork of loosely organized embryonic connectivetissue in the mesoderm from which are formed the connective tissues ofthe body, along with the blood vessels and lymphatic vessels. Vimentinis one marker of mesenchymal cells. Mesenchymal cells generally have agreater capacity to proliferate in vitro than epithelial cells and arenot fully differentiated. An “epithelial-to-mesenchymal” transition is abiological process wherein a cell, or a population of cells, from anepithelial phenotype convert to a less differentiated mesenchymalphenotype. A “mesenchymal-to-epithelial” transition is a biologicalprocess wherein a cell, or a population of cells, convert from a lessdifferentiated mesenchymal phenotype to a more differentiated epithelialphenotype.

Epitope: An antigenic determinant. These are particular chemical groupsor peptide sequences on a molecule that are antigenic (that elicit aspecific immune response). An antibody specifically binds a particularantigenic epitope on a polypeptide. Epitopes can be formed both fromcontiguous amino acids or noncontiguous amino acids juxtaposed bytertiary folding of a protein. Epitopes formed from contiguous aminoacids are typically retained on exposure to denaturing solvents whereasepitopes formed by tertiary folding are typically lost on treatment withdenaturing solvents. An epitope typically includes at least 3, and moreusually, at least 5, about 9, or 8 to 10 amino acids, and generally notmore than 20 amino acids, in a unique spatial conformation. Methods ofdetermining spatial conformation of epitopes include, for example, x-raycrystallography and 2-dimensional nuclear magnetic resonance. See, e.g.,“Epitope Mapping Protocols” in Methods in Molecular Biology, Vol. 66,Glenn E. Morris, Ed (1996). In one embodiment, an epitope binds an MHCmolecule, such as an HLA molecule or a DR molecule. These molecules bindpolypeptides having the correct anchor amino acids separated by abouteight to about ten amino acids, such as nine amino acids.

Estrogen Receptor (ER): A receptor that is activated by the hormone17β-estradiol (estrogen). The main function of the estrogen receptor isas a DNA binding transcription factor that regulates gene expression.Estrogen receptors are over-expressed in around 70% of breast cancercases, referred to as “ER positive” or “ER⁺.” Therapy for ER⁺ breastcancer involves selective estrogen receptor modulators (SERMS) whichbehave as ER antagonists in breast tissue or aromatase inhibitors. ERstatus is also used to determine sensitivity of breast cancer lesions totamoxifen and aromatase inhibitors.

Expression Control Sequences: Nucleic acid sequences that regulate theexpression of a heterologous nucleic acid sequence to which they areoperatively linked. Expression control sequences are operatively linkedto a nucleic acid sequence when the expression control sequences controland regulate the transcription and, as appropriate, translation of thenucleic acid sequence. Thus, expression control sequences can includeappropriate promoters, enhancers, transcription terminators, a startcodon (i.e., ATG) in front of a protein-encoding gene, splicing signalfor introns, maintenance of the correct reading frame of that gene topermit proper translation of mRNA, and stop codons. The term “controlsequences” is intended to include, at a minimum, components whosepresence can influence expression, and can also include additionalcomponents whose presence is advantageous, for example, leader sequencesand fusion partner sequences. Expression control sequences can include apromoter.

A promoter is a minimal sequence sufficient to direct transcription.Also included are those promoter elements which are sufficient to renderpromoter-dependent gene expression controllable for cell-type specific,tissue-specific, or inducible by external signals or agents; suchelements may be located in the 5′ or 3′ regions of the gene. Bothconstitutive and inducible promoters are included (see e.g., Bitter etal., Methods in Enzymology 153:516-544, 1987). For example, when cloningin bacterial systems, inducible promoters such as pL of bacteriophagelambda, plac, ptrp, ptac (ptrp-lac hybrid promoter) and the like can beused. In one embodiment, when cloning in mammalian cell systems,promoters derived from the genome of mammalian cells (such as themetallothionein promoter) or from mammalian viruses (such as theretrovirus long terminal repeat; the adenovirus late promoter; thevaccinia virus 7.5K promoter) can be used. Promoters are also disclosedherein that are effective when included in a poxviral vector. Promotersproduced by recombinant DNA or synthetic techniques can also be used toprovide for transcription of the nucleic acid sequences.

HER2: Human Epidermal growth factor Receptor 2 (Her2) is also known asHer 2/neu (or ErbB-2, ERBB2). It is a member of the ErbB protein family(also known as the epidermal growth factor receptor family). HER2 hasalso been designated as CD340 (cluster of differentiation 340) and p185.HER2 is notable for its role in the pathogenesis of breast cancer and asa target of treatment. It is a cell membrane surface-bound receptortyrosine kinase and is normally involved in the signal transductionpathways leading to cell growth and differentiation.

Approximately 15-20 percent of breast cancers have an amplification ofthe HER2 gene or overexpression of its protein product. Overexpressionof this receptor in breast cancer has been associated with increaseddisease recurrence and worse prognosis. Because of its prognostic role,breast tumors are routinely checked for overexpression of HER2.Overexpression also occurs in other cancer such as ovarian cancer,stomach cancer, and biologically aggressive forms of uterine cancer,such as uterine serous endometrial carcinoma.

Heterologous: Originating from separate genetic sources or species. Apolypeptide that is heterologous to Brachyury originates from a nucleicacid that does not encode Brachyury. In specific, non-limiting examples,with regard to a polypeptide comprising Brachyury, a heterologous aminoacid sequence includes a 3-galactosidase, a maltose binding protein, andalbumin, hepatitis B surface antigen, or an immunoglobulin amino acidsequence. Generally, an antibody that specifically binds to a protein ofinterest, such as Brachyury, will not specifically bind to aheterologous protein.

Host cells: Cells in which a vector can be propagated and its DNAexpressed. The cell may be prokaryotic or eukaryotic. The cell can bemammalian, such as a human cell. The term also includes any progeny ofthe subject host cell. It is understood that all progeny may not beidentical to the parental cell since there may be mutations that occurduring replication. However, such progeny are included when the term“host cell” is used.

Immune response: A response of a cell of the immune system, such as a Bcell, T cell, or monocyte, to a stimulus. In one embodiment, theresponse is specific for a particular antigen (an “antigen-specificresponse”). In one embodiment, an immune response is a T cell response,such as a CD4+ response or a CD8+ response. In another embodiment, theresponse is a B cell response, and results in the production of specificantibodies.

Immunogenic polypeptide and Immunogenic Protein: A protein or peptidewhich comprises an allele-specific motif or other sequence such that thepeptide will bind an MHC molecule and induce a T cell response, or a Bcell response (e.g. antibody production) against the antigen.

Immunogenic peptides are generally 7 to 20 amino acids in length, suchas 9 to 12 amino acids in length. In one example, an immunogenicpolypeptide includes an allele-specific motif or other sequence suchthat the peptide will bind an MHC molecule and induce a T cell responseagainst the antigen (protein) from which the immunogenic polypeptide isderived. In one embodiment, immunogenic peptides are identified usingsequence motifs or other methods, such as neural net or polynomialdeterminations, known in the art. Typically, algorithms are used todetermine the “binding threshold” of peptides to select those withscores that give them a high probability of binding at a certainaffinity and will be immunogenic. The algorithms are based either on theeffects on MHC binding of a particular amino acid at a particularposition, the effects on antibody binding of a particular amino acid ata particular position, or the effects on binding of a particularsubstitution in a motif-containing peptide. Within the context of animmunogenic peptide, a “conserved residue” is one which appears in asignificantly higher frequency than would be expected by randomdistribution at a particular position in a peptide. In one embodiment, aconserved residue is one where the MHC structure may provide a contactpoint with the immunogenic peptide. In one example, an immunogenic“Brachyury polypeptide” is a series of contiguous amino acid residuesfrom the Brachyury protein generally between 7 and 20 amino acids inlength, such as about 8 to 11 residues in length. Specific immunogenicBrachyury polypeptides are 9 or 10 amino acid residues in length, or atmost 12 amino acids in length.

Immunogenic peptides and proteins can also be identified by measuringtheir binding to a specific MHC protein (Class I or Class II) and bytheir ability to stimulate CD4 and/or CD8 when presented in the contextof the MHC protein. The characteristics of immunogenic polypeptides, aredisclosed, for example, in PCT Publication No. WO 00/12706, which isincorporated herein by reference.

Generally, an immunogenic Brachyury protein includes a number ofimmunogenic polypeptides, and can be used to induce an immune responsein a subject, such as a CD4+ T cell response. In one example, animmunogenic Brachyury protein, when bound to a Major HistocompatibilityComplex Class II molecule, activates CD4+ T cells against cellsexpressing wild-type Brachyury protein, and/or when bound to a MajorHistocompatibility Complex Class I molecule, activates cytotoxic Tlymphocytes (CTLs) against cells expressing wild-type Brachyury protein.Induction of CTLs using synthetic peptides and CTL cytotoxicity assaysare known in the art, see U.S. Pat. No. 5,662,907, which is incorporatedherein by reference.

Immunogenic composition: A composition, such as a composition comprisinga Brachyury protein or a nucleic acid encoding the Brachyury protein,that induces a measurable T cell response against cells expressingBrachyury protein, or induces a measurable B cell response (such asproduction of antibodies that specifically bind Brachyury) against aBrachyury protein. For in vitro use, the immunogenic composition canconsist of the isolated nucleic acid, vector including the nucleicacid/or immunogenic protein. For in vivo use, the immunogeniccomposition will typically comprise the nucleic acid, vector includingthe nucleic acid, and or immunogenic protein, in pharmaceuticallyacceptable carriers, and/or other agents. An immunogenic composition canoptionally include an adjuvant, a costimulatory molecule, or a nucleicacid encoding a costimulatory molecule.

Immunostimulatory molecule: Molecules that stimulate the cells of theimmune system including costimulatory molecules, cytokines andimmunostimulatory nucleic acids, such as those that include a CpG motif.

Inhibiting or treating a disease: Inhibiting a disease, such as cancergrowth, refers to inhibiting the full development of a disease. Inseveral examples, inhibiting a disease refers to lessening symptoms of acancer, such as preventing the development of paraneoplastic syndrome ina person who is known to have a cancer, or lessening a sign or symptomof the cancer or reducing cancer volume. “Treatment” refers to atherapeutic intervention that ameliorates a sign or symptom of a diseaseor pathological condition related to the disease, such as the cancer.

Isolated: An “isolated” biological component (such as a nucleic acid orprotein or organelle) has been substantially separated or purified awayfrom other biological components in the cell of the organism in whichthe component naturally occurs, i.e., other chromosomal andextra-chromosomal DNA and RNA, proteins and organelles. Nucleic acidsand proteins that have been “isolated” include nucleic acids andproteins purified by standard purification methods. The term alsoembraces nucleic acids and proteins prepared by recombinant expressionin a host cell as well as chemically synthesized nucleic acids.

Label: A detectable compound or composition that is conjugated directlyor indirectly to another molecule to facilitate detection of thatmolecule. Specific, non-limiting examples of labels include fluorescenttags, enzymatic linkages, and radioactive isotopes.

Lentiviral vector: Lentiviruses are a subclass of Retroviruses.Lentiviral vectors can integrate into the genome of non-dividing cells.This feature of Lentiviruses is unique, as other Retroviruses can infectonly dividing cells. The viral genome in the form of RNA isreverse-transcribed when the virus enters the cell to produce DNA, whichis then inserted into the genome at a random position by the viralintegrase enzyme. The vector, now called a provirus, remains in thegenome and is passed on to the progeny of the cell when it divides.Lentiviral vectors include HIV-1, HIV-2, SIV (simian immunodeficiencyvirus), EIAV (equine infectious anaemia virus), FIV (felineimmunodeficiency virus), CAEV (Caprine arthritis encephalitis virus),and VMV (Visna/maedi virus) vectors. Lentiviral vectors also encompasschimeric lentiviruses derived from at least two different lentiviruses.

Linker sequence: A linker sequence is an amino acid sequence thatcovalently links two polypeptide domains. Linker sequences can beincluded in the between the Brachyury proteins disclosed herein toprovide rotational freedom to the linked polypeptide domains. By way ofexample, in a recombinant molecule comprising two Brachyury proteins,linker sequences can be provided between them, so that the proteinscomprises Brachyury protein-linker-Brachyury protein. Linker sequences,which are generally between 2 and 25 amino acids in length, are wellknown in the art and include, but are not limited to, four glycines anda serine spacer described by Chaudhary et al., Nature 339:394-397, 1989.

Listeria: A Gram-positive bacilli. The genus Listeria currently containsseven species: L. grayi. L. innocua, L. ivanovii, L. monocytogenes, L.murrayi, L. seeligeri, and L. welshimeri. L. monocytogenes is anintracellular bacterium that has been used as a vector to deliver genesin vitro.

Lymphocytes: A type of white blood cell that is involved in the immunedefenses of the body. There are two main types of lymphocytes: B cellsand T cells.

Major Histocompatibility Complex (MHC): A generic designation meant toencompass the histocompatability antigen systems described in differentspecies, including the human leukocyte antigens (“HLA”).

Mammal: This term includes both human and non-human mammals. Similarly,the term “subject” includes both human and veterinary subjects.

Neoplasm: An abnormal cellular proliferation, which includes benign andmalignant tumors, as well as other proliferative disorders.

Oligonucleotide: A linear polynucleotide sequence of up to about 100nucleotide bases in length.

Open reading frame (ORF): A series of nucleotide triplets (codons)coding for amino acids without any internal termination codons. Thesesequences are usually translatable into a peptide.

Operably linked: A first nucleic acid sequence is operably linked with asecond nucleic acid sequence when the first nucleic acid sequence isplaced in a functional relationship with the second nucleic acidsequence. For instance, a promoter is operably linked to a codingsequence if the promoter affects the transcription or expression of thecoding sequence, such as a sequence that encodes a Brachyury protein.Generally, operably linked DNA sequences are contiguous and, wherenecessary to join two protein-coding regions, in the same reading frame.

Peptide Modifications: Brachyury proteins include synthetic embodimentsof peptides described herein. In addition, analogs (non-peptide organicmolecules), derivatives (chemically functionalized peptide moleculesobtained starting with the disclosed peptide sequences) and variants(homologs) of these proteins can be utilized in the methods describedherein. Each protein or polypeptide of this disclosure is comprised of asequence of amino acids, which may be either L- and/or D-amino acids,naturally occurring and otherwise.

Protein and polypeptides can be modified by a variety of chemicaltechniques to produce derivatives having essentially the same activityas the unmodified peptides, and optionally having other desirableproperties. For example, carboxylic acid groups of the protein, whethercarboxyl-terminal or side chain, can be provided in the form of a saltof a pharmaceutically-acceptable cation or esterified to form a C₁-C₁₆ester, or converted to an amide of formula NR₁R₂ wherein R₁ and R₂ areeach independently H or C₁-C₁₆ alkyl, or combined to form a heterocyclicring, such as a 5- or 6-membered ring. Amino groups of the peptide,whether amino-terminal or side chain, can be in the form of apharmaceutically-acceptable acid addition salt, such as the HCl, HBr,acetic, benzoic, toluene sulfonic, maleic, tartaric and other organicsalts, or can be modified to C₁-C₁₆ alkyl or dialkyl amino or furtherconverted to an amide.

Hydroxyl groups of the peptide side chains may be converted to C₁-C₁₆alkoxy or to a C₁-C₁₆ ester using well-recognized techniques. Phenyl andphenolic rings of the peptide side chains may be substituted with one ormore halogen atoms, such as fluorine, chlorine, bromine or iodine, orwith C₁-C₁₆ alkyl, C₁-C₁₆ alkoxy, carboxylic acids and esters thereof,or amides of such carboxylic acids. Methylene groups of the peptide sidechains can be extended to homologous C₂-C₄ alkylenes. Thiols can beprotected with any one of a number of well-recognized protecting groups,such as acetamide groups. Those skilled in the art will also recognizemethods for introducing cyclic structures into proteins and polypeptidesto select and provide conformational constraints to the structure thatresult in enhanced stability.

Peptidomimetic and organomimetic embodiments are envisioned, whereby thethree-dimensional arrangement of the chemical constituents of suchpeptido- and organomimetics mimic the three-dimensional arrangement ofthe peptide backbone and component amino acid side chains, resulting insuch peptido- and organomimetics of a Brachyury protein havingmeasurable or enhanced ability to generate an immune response. Forcomputer modeling applications, a pharmacophore is an idealizedthree-dimensional definition of the structural requirements forbiological activity. Peptido- and organomimetics can be designed to fiteach pharmacophore with current computer modeling software (usingcomputer assisted drug design or CADD). See Walters, “Computer-AssistedModeling of Drugs,” in Klegerman & Groves, eds., 1993, PharmaceuticalBiotechnology, Interpharm Press: Buffalo Grove, Ill., pp. 165-174 andPrinciples of Pharmacology, Munson (ed.) 1995, Ch. 102, for descriptionsof techniques used in CADD. Also included are mimetics prepared usingsuch techniques.

Pharmaceutically acceptable carriers: The pharmaceutically acceptablecarriers of use are conventional. Remington's Pharmaceutical Sciences,by E. W. Martin, Mack Publishing Co., Easton, Pa., 15th Edition (1975),describes compositions and formulations suitable for pharmaceuticaldelivery of the fusion proteins herein disclosed.

In general, the nature of the carrier will depend on the particular modeof administration being employed. For instance, parenteral formulationsusually comprise injectable fluids that include pharmaceutically andphysiologically acceptable fluids such as water, physiological saline,balanced salt solutions, aqueous dextrose, glycerol or the like as avehicle. For solid compositions (such as powder, pill, tablet, orcapsule forms), conventional non-toxic solid carriers can include, forexample, pharmaceutical grades of mannitol, lactose, starch, ormagnesium stearate. In addition to biologically neutral carriers,pharmaceutical compositions to be administered can contain minor amountsof non-toxic auxiliary substances, such as wetting or emulsifyingagents, preservatives, and pH buffering agents and the like, for examplesodium acetate or sorbitan monolaurate.

A “therapeutically effective amount” is a quantity of a composition or acell to achieve a desired effect in a subject being treated. Forinstance, this can be the amount of Brachyury protein or a vectorencoding a Brachyury protein necessary to induce an immune response,inhibit cancer growth, reduce cancer volume, prevent cancer, or tomeasurably alter outward symptoms of the cancer. When administered to asubject, a dosage will generally be used that will achieve target tissueconcentrations (for example, in lymphocytes) that has been shown toachieve an in vitro effect.

Plasmid: A DNA molecule that is separate from, and can replicateindependently of, the chromosomal DNA. They are double-stranded and, inmany cases, circular. Generally, a gene to be replicated is insertedinto copies of a plasmid containing genes that make cells resistant toparticular antibiotics and a multiple cloning site (MCS, or polylinker),which is a short region containing several commonly used restrictionsites allowing the easy insertion of DNA fragments at this location.

Poliovirus: A human enterovirus and member of the family ofPicornaviridae; the wild-type poliovirus causes poliomyelitis.Poliovirus is composed of an RNA genome and a protein capsid. Thewild-type genome is a single-stranded positive-sense RNA genome that isabout 7500 nucleotides long. The viral particle is about 30 nanometresin diameter with icosahedral symmetry.

Polynucleotide: The term polynucleotide or nucleic acid sequence refersto a polymeric form of nucleotide at least 10 bases in length. Arecombinant polynucleotide includes a polynucleotide that is notimmediately contiguous with both of the coding sequences with which itis immediately contiguous (one on the 5′ end and one on the 3′ end) inthe naturally occurring genome of the organism from which it is derived.The term therefore includes, for example, a recombinant DNA which isincorporated into a vector; into an autonomously replicating plasmid orvirus; or into the genomic DNA of a prokaryote or eukaryote, or whichexists as a separate molecule (e.g., a cDNA) independent of othersequences. The nucleotides can be ribonucleotides, deoxyribonucleotides,or modified forms of either nucleotide. The term includes single- anddouble-stranded forms of DNA.

Polypeptide: A chain of amino acids, generally greater than eight aminoacids in length, such as greater than fifteen amino acids in length,which can be post-translationally modified (e.g., glycosylation orphosphorylation) that is not the complete wild-type protein. Apolypeptide can be at least 15, at least 20, at least 30, at least 40,at least 50, at least 60, at least 70, at least 80, at least 90, atleast 100, at least 200 amino acids in length. Thus, a polypeptide canbe, for example, 20-300, 30-300, 40-300, 50-300, 60-300, 70-300, 80-300,90-300, 100-300, or 200-300 amino acids in length. In additionalembodiments, a polypeptide is 15 to 10-, 20-100, 25-100, 30-100, 35-100,40-100, 45-100, 50-100, 55-100, 60-100, 65-100, 70-100, 75-100, 80-100,85-100, 90-100 or 95-100 amino acids in length. In further embodiments apolypeptide is up to 433, 434 or 435 amino acids in length.

Protein: A chain of amino acids, generally greater than 100 amino acidsin length, that has a specific function in a cell and is a completewild-type protein or the complete wild type protein without theN-terminal methionine. A protein can be post-translationally modified.In one embodiment, the protein is a Brachyury protein.

Measles virus (Morbillivirus): A negative strand RNA virus belonging theParamyxoviridae family that causes measles. Heterologous genes can beinserted into the viral genome. The non segmented genome of measlesvirus has an anti-message polarity which results in a genomic RNA which,when purified, is not translated either in vivo or in vitro and is notinfectious.

Poxvirus: Four genera of poxviruses infect humans: orthopox, parapox,yatapox, molluscipox. Orthopox includes smallpox virus (variola),vaccinia virus, cowpox virus, and monkeypox virus. Parapox includes theorf virus, pseudocowpox, and bovine papular stomatitis virus. Yatapoxincludes tanapox virus, and yaba monkey tumor virus. Molluscipoxincludes molluscum contagiosum virus (MCV). Poxviridae viral particles(virions) are generally enveloped (external enveloped virion—EEV),though the intracellular mature virion (IMV) form of the virus, whichcontains different envelope, is also infectious.

Vaccinia virus is used as an effective tool for heterologous proteinexpression. Vaccinia virus enters cells mainly by cell fusion. Thisvirus contains three classes of genes, early, intermediate and late,that are transcribed by viral RNA polymerase and associatedtranscription factors. Vaccinia virus replicates its genome in cytoplasmof the infected cells and after late gene expression virionmorphogenesis produces intracellular mature virion (IMV) that containsenvelope, although the origin of the envelope membrane is still unknown.IMV is transported to Golgi, wherein the intracellular enveloped virus(IEV) is formed. IEV transports along microtubules to reach cellperiphery and fuse with plasma membrane to become cell-associatedenveloped virus (CEV) that triggers actin tails on cell surfaces orforms the extracellular enveloped virion (EEV), which is believed to beimportant for long range dissemination within the host organism.

A “non-poxviral vector” is a vector that is not included in the fourgenera of poxviruses.

Progesterone receptor (PR): A receptor, also known as NR3C3 (nuclearreceptor subfamily 3, group C, member 3), that is a steroid receptorthat specifically binds progesterone. The progesterone receptor is notexpressed on triple negative basal breast cancer cells.

Probes and primers: A probe comprises an isolated nucleic acid attachedto a detectable label or reporter molecule. Primers are short nucleicacids, preferably DNA oligonucleotides, of about 15 nucleotides or morein length. Primers may be annealed to a complementary target DNA strandby nucleic acid hybridization to form a hybrid between the primer andthe target DNA strand, and then extended along the target DNA strand bya DNA polymerase enzyme. Primer pairs can be used for amplification of anucleic acid sequence, for example by polymerase chain reaction (PCR) orother nucleic-acid amplification methods known in the art. One of skillin the art will appreciate that the specificity of a particular probe orprimer increases with its length. Thus, for example, a primer comprising20 consecutive nucleotides will anneal to a target with a higherspecificity than a corresponding primer of only 15 nucleotides. Thus, inorder to obtain greater specificity, probes and primers can be selectedthat comprise about 20, 25, 30, 35, 40, 50 or more consecutivenucleotides.

Purified: Brachyury proteins and nucleic acids as disclosed herein canbe purified (and/or synthesized) by any of the means known in the art(see, e.g., Guide to Protein Purification, ed. Deutscher, Meth. Enzymol.185, Academic Press, San Diego, 1990; and Scopes, Protein Purification:Principles and Practice, Springer Verlag, New York, 1982). Substantialpurification denotes purification from other proteins, nucleic acids, orcellular components. The term purified does not require absolute purity;rather, it is intended as a relative term. A substantially purifiedprotein is at least about 60%, 70%, 80%, 90%, 95%, 98% or 99% pure.Thus, in one specific, non-limiting example, a substantially purifiedprotein is at least 90% free of other proteins or cellular components.In additional embodiments, a nucleic acid or cell preparation ispurified such that the nucleic acid or cell represents at least about60% (such as, but not limited to, 70%, 80%, 90%, 95%, 98% or 99%) of thetotal nucleic acid or cell content of the preparation, respectively.Thus, in one specific, non-limiting example, a substantially purifiednucleic acid is at least 90% free of other nucleic acids or cellularcomponents.

Recombinant: A recombinant nucleic acid is one that has a sequence thatis not naturally occurring or has a sequence that is made by anartificial combination of two otherwise separated segments of sequence.This artificial combination is often accomplished by chemical synthesisor, more commonly, by the artificial manipulation of isolated segmentsof nucleic acids, e.g., by genetic engineering techniques.

Replication defective: A viral vector that cannot further replicate andpackage its genomes. In one non-limiting example, when the cells of asubject are infected with a vector, a heterologous in the vector isexpressed in the subject's cells, however, due to the fact that thepatient's cells lack essential genes. Examples are the rev and cap genesfor AAV, or gag, pol and env for a lentivirus. Generally, the genesnecessary to replicate and package are not present, such that andwild-type virus cannot be formed in the subject's cells.

Salmonella: A genus of rod-shaped, Gram-negative, non-spore-forming,predominantly motile enterobacteria with diameters around 0.7 to 1.5 μm,lengths from 2 to 5 μm, and flagella which grade in all directions (i.e.peritrichous). They are chemoorganotrophs, obtaining their energy fromoxidation and reduction reactions using organic sources, and arefacultative anaerobes. Salmonella can be used as delivery vector fortherapeutic proteins, by including plasmids, such as those withtruncated tetA genes in the host cell. Attenuated S. typhimurium can betransformed with DNA plasmids, such as, but not limited to, pIRES(Invitrogen) and used as a carrier for delivery of polypeptides andproteins.

Selectively hybridize: Hybridization under moderately or highlystringent conditions that excludes non-related nucleotide sequences.

In nucleic acid hybridization reactions, the conditions used to achievea particular level of stringency will vary, depending on the nature ofthe nucleic acids being hybridized. For example, the length, degree ofcomplementarity, nucleotide sequence composition (for example, GC v. ATcontent), and nucleic acid type (for example, RNA versus DNA) of thehybridizing regions of the nucleic acids can be considered in selectinghybridization conditions. An additional consideration is whether one ofthe nucleic acids is immobilized, for example, on a filter.

A specific example of progressively higher stringency conditions is asfollows: 2×SSC/0.1% SDS at about room temperature (hybridizationconditions); 0.2×SSC/0.1%/SDS at about room temperature (low stringencyconditions); 0.2×SSC/0.1% SDS at about 42° C. (moderate stringencyconditions); and 0.1×SSC at about 68° C. (high stringency conditions).One of skill in the art can readily determine variations on theseconditions (e.g., Molecular Cloning: A Laboratory Manual, 2nd ed., vol.1-3, ed. Sambrook et al., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989). Washing can be carried out using only one ofthese conditions, e.g., high stringency conditions, or each of theconditions can be used, e.g., for 10-15 minutes each, in the orderlisted above, repeating any or all of the steps listed. However, asmentioned above, optimal conditions will vary, depending on theparticular hybridization reaction involved, and can be determinedempirically.

Sequence identity: The similarity between amino acid sequences isexpressed in terms of the similarity between the sequences, otherwisereferred to as sequence identity. Sequence identity is frequentlymeasured in terms of percentage identity (or similarity or homology);the higher the percentage, the more similar the two sequences are.Homologs or variants of a Brachyury protein will possess a relativelyhigh degree of sequence identity when aligned using standard methods.

Methods of alignment of sequences for comparison are well known in theart. Various programs and alignment algorithms are described in: Smithand Waterman, Adv. Appl. Math. 2:482, 1981; Needleman and Wunsch, J.Mol. Biol. 48:443, 1970; Higgins and Sharp, Gene 73:237, 1988; Higginsand Sharp, CABIOS 5:151, 1989; Corpet et al., Nucleic Acids Research16:10881, 1988; and Pearson and Lipman, Proc. Natl. Acad. Sci. USA85:2444, 1988. Altschul et al., Nature Genet. 6:119, 1994, presents adetailed consideration of sequence alignment methods and homologycalculations.

The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J.Mol. Biol. 215:403, 1990) is available from several sources, includingthe National Center for Biotechnology Information (NCBI, Bethesda, Md.)and on the internet, for use in connection with the sequence analysisprograms blastp, blastn, blastx, tblastn and tblastx. A description ofhow to determine sequence identity using this program is available onthe NCBI website on the internet.

Homologs and variants of a Brachyury protein are typically characterizedby possession of at least 75%, for example at least 80%, sequenceidentity, or at least 90% sequence identity, counted over the fulllength alignment with the amino acid sequence of Brachyury using theNCBI Blast 2.0, gapped blastp set to default parameters. For comparisonsof amino acid sequences of greater than about 30 amino acids, the Blast2 sequences function is employed using the default BLOSUM62 matrix setto default parameters, (gap existence cost of 11, and a per residue gapcost of 1). When aligning short peptides (fewer than around 30 aminoacids), the alignment should be performed using the Blast 2 sequencesfunction, employing the PAM30 matrix set to default parameters (open gap9, extension gap 1 penalties). Proteins with even greater similarity tothe reference sequences will show increasing percentage identities whenassessed by this method, such as at least 80%, at least 85%, at least90%, at least 95%, at least 98%, or at least 99% sequence identity. Whenless than the entire sequence is being compared for sequence identity,homologs and variants will typically possess at least 80% sequenceidentity over short windows of 10-20 amino acids, and can possesssequence identities of at least 85% or at least 90% or 95% depending ontheir similarity to the reference sequence. Methods for determiningsequence identity over such short windows are available at the NCBIwebsite on the internet. One of skill in the art will appreciate thatthese sequence identity ranges are provided for guidance only; it isentirely possible that strongly significant homologs could be obtainedthat fall outside of the ranges provided.

Specific binding agent: An agent that binds substantially only to adefined target. Thus a Brachyury specific binding agent is an agent thatbinds substantially to a Brachyury protein. In one embodiment, thespecific binding agent is a monoclonal or polyclonal antibody thatspecifically binds Brachyury protein.

T Cell: A white blood cell critical to the immune response. T cellsinclude, but are not limited to, CD4⁺ T cells and CD8⁺ T cells. A CD4⁺ Tlymphocyte is an immune cell that carries a marker on its surface knownas “cluster of differentiation 4” (CD4) and is MHC Class II restricted.These cells, often called helper T cells, help orchestrate the immuneresponse, including antibody responses as well as killer T cellresponses. CD8⁺ T cells carry the “cluster of differentiation 8” (CD8)marker and are MHC Class I restricted. In one embodiment, a CD8 T cellis a cytotoxic T lymphocyte. In another embodiment, a CD8 cell is asuppressor T cell.

Therapeutically active protein: An agent composed of amino acids, suchas a Brachyury protein, that causes induction of an immune response, asmeasured by clinical response (for example increase in a population ofimmune cells, increased cytolytic activity against cells that expressBrachyury, or measurable reduction of tumor burden). Therapeuticallyactive molecules can also be made from nucleic acids. Examples of anucleic acid based therapeutically active molecule is a nucleic acidsequence that encodes a Brachyury protein, wherein the nucleic acidsequence is operably linked to a control element such as a promoter.

In one embodiment, a therapeutically effective amount of a composition,such as a Brachyury protein or a vector encoding the Brachyury protein,is an amount used to generate an immune response, or to treat or preventcancer in a subject. In several examples, “treatment” refers to atherapeutic intervention that ameliorates a sign or symptom of a cancer,or a reduction in tumor burden.

Transduced: A transduced cell is a cell into which has been introduced anucleic acid molecule by molecular biology techniques. As used herein,the term transduction encompasses all techniques by which a nucleic acidmolecule might be introduced into such a cell, including transfectionwith viral vectors, transformation with plasmid vectors, andintroduction of naked DNA by electroporation, lipofection, and particlegun acceleration.

Vector: A nucleic acid molecule as introduced into a host cell, therebyproducing a transformed host cell. A vector may include nucleic acidsequences that permit it to replicate in a host cell, such as an originof replication. A vector may also include one or more selectable markergene and other genetic elements known in the art. Vectors includeplasmid vectors, including plasmids for expression in gram negative andgram positive bacterial cell. Exemplary vectors include those forexpression in E. coli and Salmonella. Vectors also include poxviralvectors, such as, but are not limited to, retrovirus, orthopox, avipox,fowlpox, capripox, suipox, adenoviral, herpes virus, alpha virus,baculovirus, Sindbis virus, vaccinia virus and poliovirus vectors.Vectors also include vectors for expression in yeast cells.

Yeast: Unicellular microorganisms that belong to one of three classes:Ascomycetes, Basidiomycetes and Fungi Imperfecti. A yeast can be anon-pathogenic strain such as Saccharomyces cerevisiae. Yeast strainsinclude Saccharomyces, Candida (which can be pathogenic), Cryptococcus,Hansenula, Kluyveromyces, Pichia, Rhodotorula, Schizosaccharomyces andYarrowia. Yeast genera include Saccharomyces, Candida, Hansenula. Pichiaor Schizosaccharomyces. Species of yeast strains include Saccharomycescerevisiae, Saccharomyces carlsbergensis, Candida albicans, Candidakefyr, Candida tropicalis, Cryptococcus laurentii, Cryptococcusneoformans, Hansenula anomala, Hansenula polymorpha, Kluyveromycesfragilis, Kluyveromyces lactis, Kluyveromyces marxianus var. lactis,Pichia pastoris. Rhodotorula rubra, Schizosaccharomyces pombe, andYarrowia lipolytica.

Yeast vehicles” include, but are not limited to, a live intact (whole)yeast microorganism (i.e., a yeast cell having all its componentsincluding a cell wall), a killed (dead) or inactivated intact yeastmicroorganism, or derivatives of intact yeast including: a yeastspheroplast (i.e., a yeast cell lacking a cell wall), a yeast cytoplast(i.e., a yeast cell lacking a cell wall and nucleus), a yeast ghost(i.e., a yeast cell lacking a cell wall, nucleus and cytoplasm), asubcellular yeast membrane extract or fraction thereof (also referred toas a yeast membrane particle or a subcellular yeast particle), any otheryeast particle, or a yeast cell wall preparation. A “non-yeast vector”is a composition that does not include yeast vehicles.

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this disclosure belongs. The singular terms“a,” “an,” and “the” include plural referents unless context clearlyindicates otherwise. The term “comprises” means “includes.” Similarly,comprising “A or B” includes “A,” “B,” and both “A and B.” It is furtherto be understood that all base sizes or amino acid sizes, and allmolecular weight or molecular mass values, given for nucleic acids orpolypeptides are approximate, and are provided for description. Althoughmethods and materials similar or equivalent to those described hereincan be used in the practice or testing of this disclosure, suitablemethods and materials are described below. All publications, patentapplications, patents, and other references mentioned herein areincorporated by reference in their entirety. In case of conflict, thepresent specification, including explanations of terms, will control. Inaddition, the materials, methods, and examples are illustrative only andnot intended to be limiting.

Immunogenic Brachyury Protein and Brachyury Polypeptides

Brachyury (also known as “T-protein”) is a protein which is transcribedin the mesoderm. In one embodiment, the Brachyury protein has a sequenceset forth as:

(SEQ ID NO: 1) MSSPGTESAGKSLQYRVDHLLSAVENELQAGSEKGDPTERELRVGLEESELWLRFKELTNEMIVTKNGRRMFPVLKVNVSGLDPNAMYSFLLDFVAADNHRWKYVNGEWVPGGKPEPQAPSCVYIHPDSPNFGAHWMKAPVSFSKVKLTNKLNGGGQIMLNSLHKYEPRIHIVRVGGPQRMITSHCFPETQFIAVTAYQNEEITALKIKYNPFAKAFLDAKERSDHKEMMEEPGDSQQPGYSQWGWLLPGTSTLCPPANPHPQFGGALSLPSTHSCDRYPTLRSHRSSPYPSPYAHRNNSPTYSDNSPACLSMLQSHDNWSSLGMPAHPSMLPVSHNASPPTSSSQYPSLWSVSNGAVTPGSQAAAVSNGLGAQFFRGSPAHYTPLTHPVSAPSSSGSPLYEGAAAATDIVDSQYDAAAQGRL IASWTPVSPPSM(see also GENBANK ® Accession No NP_003172 andGENBANK ® Accession No. NM_003181, as availableon Feb. 23, 2007, incorporated herein by reference).

Using the genetic code, one of skill in the art can readily produce anucleic acid sequence encoding Brachyury. In one example, Brachyuryprotein is encoded by a nucleic acid having a sequence set forth as:

(SEQ ID NO: 2) tttgcttttg cttatttccg tccatttccc tctctgcgcgcggaccttcc ttttccagat ggtgagagcc gcggggacacccgacgccgg ggcaggctga tccacgatcc tgggtgtgcgtaacgccgcc tggggctccg tgggcgaggg acgtgtggggacaggtgcac cggaaactgc cagactggag agttgaggcatcggaggcgc gagaacagca ctactactgc ggcgagacgagcgcggcgca tcccaaagcc cggccaaatg cgctcgtccctgggagggga gggaggcgcg cctggagcgg ggacagtcttggtccgcgcc ctcctcccgg gtctgtgccg ggacccgggacccgggagcc gtcgcaggtc tcggtccaag gggccccttttctcggaagg gcggcggcca agagcaggga aggtggatctcaggtagcga gtctgggctt cggggacggc ggggaggggagccggacggg aggatgagct cccctggcac cgagagcgcgggaaagagcc tgcagtaccg agtggaccac ctgctgagcgccgtggagaa tgagctgcag gcgggcagcg agaagggcgaccccacagag cgcgaactgc gcgtgggcct ggaggagagcgagctgtggc tgcgcttcaa ggagctcacc aatgagatgatcgtgaccaa gaacggcagg aggatgtttc cggtgctgaaggtgaacgtg tctggcctgg accccaacgc catgtactccttcctgctgg acttcgtggc ggcggacaac caccgctggaagtacgtgaa cggggaatgg gtgccggggg gcaagccggagccgcaggcg cccagctgcg tctacatcca ccccgactcgcccaacttcg gggcccactg gatgaaggct cccgtctccttcagcaaagt caagctcacc aacaagctca acggagggggccagatcatg ctgaactcct tgcataagta tgagcctcgaatccacatag tgagagttgg gggtccacag cgcatgatcaccagccactg cttccctgag acccagttca tagcggtgactgcttatcag aacgaggaga tcacagctct taaaattaagtacaatccat ttgcaaaagc tttccttgat gcaaaggaaagaagtgatca caaagagatg atggaggaac ccggagacagccagcaacct gggtactccc aatgggggtg gcttcttcctggaaccagca ccctgtgtcc acctgcaaat cctcatcctcagtttggagg tgccctctcc ctcccctcca cgcacagctgtgacaggtac ccaaccctga ggagccaccg gtcctcaccctaccccagcc cctatgctca tcggaacaat tctccaacctattctgacaa ctcacctgca tgtttatcca tgctgcaatcccatgacaat tggtccagcc ttggaatgcc tgcccatcccagcatgctcc ccgtgagcca caatgccagc ccacctaccagctccagtca gtaccccagc ctgtggtctg tgagcaacggcgccgtcacc ccgggctccc aggcagcagc cgtgtccaacgggctggggg cccagttctt ccggggctcc cccgcgcactacacacccct cacccatccg gtctcggcgc cctcttcctcgggatcccca ctgtacgaag gggcggccgc ggccacagacatcgtggaca gccagtacga cgccgcagcc caaggccgcctcatagcctc atggacacct gtgtcgccac cttccatgtgaagcagcaag gcccaggtcc cgaaagatgc agtgactttttgtcgtggca gccagtggtg actggattga cctactaggtacccagtggc agtctcaggt taagaaggaa atgcagcctcagtaacttcc ttttcaaagc agtggaggag cacacggcacctttccccag agccccagca tcccttgctc acacctgcagtagcggtgct gtcccaggtg gcttacagat gaacccaactgtggagatga tgcagttggc ccaacctcac tgacggtgaaaaaatgtttg ccagggtcca gaaacttttt ttggtttatttctcatacag tgtattggca actttggcac accagaatttgtaaactcca ccagtcctac tttagtgaga taaaaagcacactcttaatc ttcttccttg ttgctttcaa gtagttagagttgagctgtt aaggacagaa taaaatcata gttgaggacagcaggtttta gttgaattga aaatttgact gctctgccccctagaatgtg tgtattttaa gcatatgtag ctaatctcttgtgttgttaa actataactg tttcatattt ttcttttgacaaagtagcca aagacaatca gcagaaagca ttttctgcaaaataaacgca atatgcaaaa tgtgattcgt ccagttattagtgaagcccc tccttttgtg agtatttact gtttattg.

In other embodiments, Brachyury protein has an amino acid sequence atleast 90% identical to SEQ ID NO: 1, for example a polypeptide that isat least or about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%identical to SEQ ID NO: 1. Brachyury proteins are disclosed herein thatcan be used to induce an immune response (are immunogenic), wherein theBrachyury protein can produce a Brachyury-specific CD4+ T cell response.In some embodiments, the Brachyury protein produces a Brachyury specificCD4+ T cell response and a Brachyury Specific CD8+ T cell response.

SEQ ID NO: 1 provides an exemplary sequence for the full-lengthBrachyury; another full length Brachyury is this amino acid sequencewith the N-terminal methionine removed. In some examples, the Brachyuryprotein includes the amino acid sequence set forth as SEQ ID NO: 1, withsubstitutions at position 177 (Asp vs. Gly, respectively), position 368(Thr vs. Ser, respectively) and position 409 (Asn vs. Asp,respectively). Thus, these sequences can be used to induce a Brachyuryspecific CD4+ T cell response.

Positions 41 to 223 of the amino acid sequence set forth as SEQ ID NO: 1represents the T-box DNA binding domain of human Brachyury, and theT-box domain in other Brachyury sequences, including Brachyury sequencesfrom other species, can be readily identified by comparison to thesesequences. As used herein, reference to a T-box domain of a Brachyuryprotein can include an additional 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 consecutive amino acids of theBrachyury sequence on the N-terminal and/or the C-terminal end of thedefined T-box domain (e.g., on either side of positions 41-223 of SEQ IDNO: 1). In some embodiments, the Brachyury protein comprises the T-boxdomain of SEQ ID NO: 1 and induces a Brachyury specific CD4+ T cellresponse. In some embodiment, the polypeptide includes the T-box DNAbinding domain or a portion thereof.

Human Brachyury has very high homology with Brachyury from other animalspecies and therefore, the sequences of Brachyury from other organismscan be utilized, particularly where these sequences are identical,substantially homologous, and elicit an effective immune responseagainst the target antigen (e.g., native Brachyury expressed by a tumorcell). For example, murine Brachyury, which was cloned by Hermann andcolleagues in 1990 (Hermann et al., supra), and is approximately 85%identical to human Brachyury at the nucleotide level. Murine Brachyuryis approximately 91% identical to human Brachyury at the amino acidlevel. With respect to Brachyury from other animals, at the amino acidlevel, human Brachyury is 99.5% identical to Brachyury from Pantroglodytes, 90.1% identical to Brachyury from Canis lupus familiaris,88.5% identical to Brachyury from Bos Taurus, 92.2% identical toBrachyury from Rattus norvegicus, and 80.9% identical to Brachyury fromGallus gallus. Nucleic acids encoding these Brachyury proteins can beused in the poxviral vectors and methods disclosed herein. Generally,the T-box domain of these Brachyury proteins is included in the regionof amino acids 1-223. These polypeptides can be used to induce aBrachyury specific CD4+ T cell response.

Mouse and human Brachyury differ by two amino acids (at positions 26 and96) in the T-box region. The murine Brachyury has the amino acidsequence set forth as:

(SEQ ID NO: 3, 436 amino acids)MSSPGTESAGKSLQYRVDHLLSAVESELQAGSEKGDPTERELRVGLEESELWLRFKELTNEMIVTKNGRRMFPVLKVNVSGLDPNAMYSFLLDFVTADNHRWKYVNGEWVPGGKPEPQAPSCVYIHPDSPNFGAHWMKAPVSFSKVKLTNKLNGGGQIMLNSLHKYEPRIHIVRVGGPQRMITSHCFPETQFIAVTAYQNEEITALKIKYNPFAKAFLDAKERNDHKDVMEEPGDCQQPGYSQWGWLVPGAGTLCPPASSHPQFGGSLSLPSTHGCERYPALRNHRSSPYPSPYAHRNSSPTYADNSSACLSMLQSHDNWSSLGVPGHTSMLPVSHNASPPTGSSQYPSLWSVSNGTITPGSQTAGVSNGLGAQFFRGSPAHYTPLTHTVSAATSSSSGSPMYEGAATVTDISDSQYDTAQSL LIASWTPVSPPSMA nucleotide sequence encoding murine Brachyury is:

(SEQ ID NO: 4) ggctccgcag agtgaccctt tttcttggaa aagcggtggcgagagaagtg aaggtggctg ttgggtaggg agtcaagactcctggaaggt ggagagggtg gcgggaggatgagctcgccgggcacagaga gcgcagggaa gagcctgcagtaccgagtgg accacctgct cagcgccgtggagagcgagctgcaggcggg cagcgagaag ggagacccca ccgaacgcgaactgcgagtg ggcctggagg agagcgagct gtggctgcgcttcaaggagc taactaacga gatgattgtg accaagaacggcaggaggat gttcccggtg ctgaaggtaa atgtgtcaggcctggacccc aatgccatgt actctttctt gctggacttcgtgacggctg acaaccaccg ctggaaatat gtgaacggggagtgggtacc tgggggcaaa ccagagcctc aggcgcccagctgcgtctac atccacccag actcgcccaa ttttggggcccactggatga aggcgcctgt gtctttcagc aaagtcaaactcaccaacaa gctcaatgga gggggacaga tcatgttaaactccttgcat aagtatgaac ctcggattca catcgtgagagttgggggcc cgcaacgcat gatcaccagc cactgctttcccgagaccca gttcatagct gtgactgcct accagaatgaggagattaca gcccttaaaa ttaaatacaa cccatttgctaaagccttcc ttgatgccaa agaaagaaac gaccacaaagatgtaatgga ggaaccgggg gactgccagc agccggggtattcccaatgg gggtggcttg ttcctggtgc tggcaccctctgcccgcctg ccagctccca ccctcagttt ggaggctcgctctctctccc ctccacacac ggctgtgaga ggtacccagctctaaggaac caccggtcat cgccctaccc cagcccctatgctcatcgga acagctctcc aacctatgcg gacaattcatctgcttgtct gtccatgctg cagtcccatg ataactggtctagcctcgga gtgcctggcc acaccagcat gctgcctgtgagtcataacg ccagcccacc tactggctct agccagtatcccagtctctg gtctgtgagc aatggtacca tcaccccaggctcccagaca gctggggtgt ccaacgggct gggagctcagttctttcgag gctcccctgc acattacaca ccactgacgcacacggtctc agctgccacg tcctcgtctt ctggttctccgatgtatgaa ggggctgcta cagtcacaga catttctgacagccagtatg acacggccca aagcctcctc atagcctcgtggacacctgt gtcaccccca tctatgtgaa ttgaactttcctccatgtgc tgagacttgt aacaaccggt gtcaactggatcttctaggc tcaaagtggc aggctcttgg gacaagggaaaaataaataa ataaaagcta gatactaaca actccattttcaaataagag caataataca tgtcctataa tcatgttctacagcctcttg tttgatacct acagtagtga tatgtgtcctacattatgaa gccaaggaca gagagacggc tgtggtccagttttttgtga ctggcagtta atcagagtcc tttgctaggtagggtcctat atcttgtgtt tctctacaac atatatgtgactttgaaatc ctggaattcg tccaccccct gtcctactttagtgagacac aaggtacacc tctaatgtcc tcccttgttgccttagagta gttaactttg aggacagaaa aaagcatagccagaagattg taactgaacc gtcaactgtt ctgcccttggaacatgccta ctttaagcac acgtagcttt ttgtgttgggaagtcaactg tatggatact tttctgttga caaagtagccaaagacaatc tgcagaaagt gttttctgca caataaaggc aatatatagc acctgg,See also the amino acid and nucleic acidsequences set forth in GENBANK ®Accession No. NM_009309 (GI: 118130357),Oct. 29, 2011, incorporated herein by reference.Positions 41 to 223 of SEQ ID NO:4 represent the T-box DNA bindingdomain of murine Brachyury. These Brachyury proteins can also be used toinduce a Brachyury specific T cell response.

In one embodiment, the Brachyury protein includes, consists essentiallyof, or consists of, an amino acid sequence at least at least 90%identical to SEQ ID NO: 1, for example a polypeptide that is about 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 1. Inanother embodiment, the Brachyury proteins do not include the firstamino acid of SEQ ID NO: 1 (methionine). IN further embodiments, theBrachyury protein includes, or consists of, amino acids 2-435 of SEQ IDNO: 1. In yet another embodiment, the Brachyury protein includes, orconsists of, the amino acid sequence set forth as SEQ ID NO:1, withsubstitutions at position 177 (Asp vs. Gly, respectively), position 368(Thr vs. Ser, respectively) and position 409 (Asn vs. Asp,respectively).

In some examples, the Brachyury protein includes amino acids 1-15 of SEQID NO: 1. In yet another embodiment, the Brachyury protein includes,consists essentially of, or consists of, the amino acid sequence setforth as SEQ ID NO: 3.

Brachyury polypeptides are also of use in the methods disclosed herein.The These Brachyury polypeptides include at least 15, at least 20, atleast 30, at least 40, at least 50, at least 60, at least 70, at least80, at least 90, at least 100, at least 200, or at least 300, or atleast 400 amino acids of a Brachyury protein, such as 435 amino acids ofa Brachyury protein. The Brachyury protein can include 15, 20, 30, 40,50, 60, 70, 80, 90, 100, 200, 300 or 400 amino acids of a Brachyuryprotein. In some embodiments, a Brachyury polypeptide is 15-400, 20-400,30-400, 40-400, 50-400, 60-400, 70-400, 80-400, 90-400, 100-400, or200-400 amino acids of a Brachyury protein. In other embodiments, aBrachyury polypeptide is 15-300, 20-300, 30-300, 40-300, 50-300, 60-300,70-300, 80-300, 90-300, 100-300, or 200-300 amino acids of a Brachyuryprotein. In additional embodiments, the Brachyury polypeptide is 15 to10-, 20-100, 25-100, 30-100, 35-100, 40-100, 45-100, 50-100, 55-100,60-100, 65-100, 70-100, 75-100, 80-100, 85-100, 90-100 or 95-100 aminoacids of any of the Brachyury proteins disclosed herein. The Brachyurypolypeptide can be 15, 50, 100, 150, 250, 300, 350, 400, 430, 431, 432,433 or 434 amino acids in length. The Brachyury polypeptide can be15-430, 15-431, 15-432, 15-433, 15-434 or 15-435 amino acids in length.An exemplary polypeptide is shown in FIG. 2.

In further embodiments, a Brachyury polypeptide is 15-20 amino acids inlength, such as 15-17 amino acids in length, such as 15, 16, 17, 17, 19or 20 amino acids in length and binds MHC Class II. The MHC Class IIantigen can be encoded by a HLA-DP, HLA-DR, HLA-B, HLA-DQA1 or HLA-DQB1allele.

It is disclosed herein that Brachyury protein, Brachyury polypeptides,nucleic acids encoding Brachyury proteins and polypeptides, and non-poxnon-yeast viral vectors including a polynucleotide encoding a Brachyuryprotein can be used to induce Brachyury specific CD4+ T cells in asubject. In additional embodiments the Brachyury protein, Brachyurypolypeptide, polynucleotide encoding a Brachyury protein or polypeptide,or non-pox non-yeast vector include the polynucleotide induce aBrachyury specific CD8+ T cell response, or both a Brachyury specificCD4+ T cell response and a CD8+ T cell response.

In several embodiments, the isolated Brachyury protein or polypeptide isincluded in a fusion protein. Thus, the fusion protein can include theBrachyury protein or Brachyury polypeptide (see above) and a secondheterologous moiety, such as a myc protein, an enzyme or a carrier (suchas a hepatitis carrier protein or bovine serum albumin) covalentlylinked to the Brachyury protein or polypeptide. Thus, in severalspecific non-limiting examples, the fusion protein includes a Brachyuryprotein (or Brachyury polypeptide) and six sequential histidineresidues, a β-galactosidase amino acid sequence, and/or animmunoglobulin amino acid sequence.

Brachyury proteins or polypeptides that are linked to a carrier are alsoof use in the disclosed methods. Generally, a carrier is an immunogenicmacromolecule to which an antigenic molecule can be bound. When bound toa carrier, the bound Brachyury protein or Brachyury polypeptide becomesmore immunogenic. Carriers are chosen to increase the immunogenicity ofthe bound molecule and/or to elicit higher titers of antibodies againstthe carrier which are diagnostically, analytically, and/ortherapeutically beneficial. Covalent linking of a molecule to a carriercan confer enhanced immunogenicity and T cell dependence (see Pozsgay etal., PNAS 96:5194-97, 1999; Lee et al., J. Immunol. 116:1711-18, 1976;Dintzis et al., PNAS 73:3671-75, 1976). Useful carriers includepolymeric carriers, which can be natural (for example, polysaccharides,polypeptides or proteins from bacteria or viruses), semi-synthetic orsynthetic materials containing one or more functional groups to which areactant moiety can be attached. Bacterial products and viral proteins(such as hepatitis B surface antigen and core antigen) can also be usedas carriers, as well as proteins from higher organisms such as keyholelimpet hemocyanin, horseshoe crab hemocyanin, edestin, mammalian serumalbumins, and mammalian immunoglobulins. Suitable carriers include, butare not limited to, a hepatitis B small envelope protein HBsAg. Thisprotein has the capacity to self-assemble into aggregates and can formviral-like particles. The preparation of HBsAg is well documented, seefor example European Patent Application Publication No. EP-A-0 226 846,European Patent Application Publication No. EP-A-0 299 108 and PCTPublication No. WO 01/117554, and the amino acid sequence disclosed, forexample, in Tiollais et al., Nature, 317: 489, 1985, and European PatentPublication No. EP-A-0 278 940, and PCT Publication No. WO 91/14703, allof which are incorporated herein by reference.

In other embodiments, only the Brachyury protein or polypeptide isutilized. Thus, a second heterologous moiety is non-covalently linked tothe Brachyury protein or polypeptide.

Nucleic Acids Encoding Brachyury Protein and Polypeptides

Nucleic acids that encode a Brachyury protein and/or polypeptide canreadily be produced. These nucleic acids include DNA, cDNA and RNAsequences which encode the Brachyury polypeptide of interest. Silentmutations in the coding sequence result from the degeneracy (i.e.,redundancy) of the genetic code, whereby more than one codon can encodethe same amino acid residue. Thus, for example, leucine can be encodedby CTT, CTC, CTA, CTG, TTA, or TTG; serine can be encoded by TCT, TCC,TCA, TCG, AGT, or AGC; asparagine can be encoded by AAT or AAC; asparticacid can be encoded by GAT or GAC; cysteine can be encoded by TGT orTGC; alanine can be encoded by GCT, GCC, GCA, or GCG; glutamine can beencoded by CAA or CAG; tyrosine can be encoded by TAT or TAC; andisoleucine can be encoded by ATT, ATC, or ATA. Tables showing thestandard genetic code can be found in various sources (e.g., L. Stryer,1988, Biochemistry, 3.sup.rd Edition, W.H. 5 Freeman and Co., NY).

A nucleic acid encoding a Brachyury protein can be cloned or amplifiedby in vitro methods, such as the polymerase chain reaction (PCR), theligase chain reaction (LCR), the transcription-based amplificationsystem (TAS), the self-sustained sequence replication system (3SR) andthe Qβ replicase amplification system (QB). For example, apolynucleotide encoding the Brachyury protein can be isolated bypolymerase chain reaction of cDNA using primers based on the DNAsequence of the molecule. A wide variety of cloning and in vitroamplification methodologies are well known to persons skilled in theart. PCR methods are described in, for example, U.S. Pat. No. 4,683,195;Mullis et al., Cold Spring Harbor Symp. Quant. Biol. 51:263, 1987; andErlich, ed., PCR Technology, (Stockton Press, N Y, 1989).Polynucleotides also can be isolated by screening genomic or cDNAlibraries with probes selected from the sequences of the desiredpolynucleotide under stringent hybridization conditions.

A polynucleotide sequence encoding a Brachyury protein or polypeptidecan be operatively linked to expression control sequences. An expressioncontrol sequence operatively linked to a coding sequence is ligated suchthat expression of the coding sequence is achieved under conditionscompatible with the expression control sequences. The expression controlsequences include, but are not limited to, appropriate promoters,enhancers, transcription terminators, a start codon (i.e., ATG) in frontof a protein-encoding gene, splicing signal for introns, maintenance ofthe correct reading frame of that gene to permit proper translation ofmRNA, and stop codons. Suitable promoters include, but are not limitedto, an SV40 early promoter, RSV promoter, adenovirus major latepromoter, human CMV immediate early I promoter, poxvirus promoter, 30Kpromoter, I3 promoter, sE/L promoter, 7.5K promoter, 40K promoter, andC1 promoter. T DNA vaccines are described in U.S. Pat. Nos. 5,589,466;5,973,972, which are each incorporated herein by reference. In additionto the delivery protocols described in those applications, alternativemethods of delivering DNA are described in U.S. Pat. Nos. 4,945,050 and5,036,006, which are both incorporated herein by reference.

Plasmids have been designed with a number of goals in mind, such asachieving regulated high copy number and avoiding potential causes ofplasmid instability in bacteria, and providing means for plasmidselection that are compatible with human therapeutic use. Particularattention has been paid to the dual requirements of gene therapyplasmids. First, they are suitable for maintenance and fermentation inE. coli, so that large amounts of DNA can be produced and purified.Second, they are safe and suitable for use in human patients andanimals. The first requirement calls for high copy number plasmids thatcan be selected for and stably maintained relatively easily duringbacterial fermentation. The second requirement calls for attention toelements such as selectable markers and other coding sequences. In someembodiments plasmids that encode a Brachyury protein or polypeptide arecomposed of: (1) a high copy number replication origin, (2) a selectablemarker, such as, but not limited to, the neo gene for antibioticselection with kanamycin, (3) transcription termination sequences, and(4) a multicloning site for incorporation of various nucleic acidcassettes; and (5) a nucleic acid sequence encoding a Brachyury proteinand/or a Brachyury polypeptide.

There are numerous plasmid vectors that are known in the art forinducing a nucleic acid encoding a protein. These include, but are notlimited to, the vectors disclosed in U.S. Pat. Nos. 6,103,470;7,598,364; 7,989,425; and 6,416,998, which are incorporated herein byreference.

Non-Pox Non-Yeast Vectors

Non-poxviral non-yeast vectors can be used to express the Brachyuryproteins and/or polypeptides disclosed herein. These vectors are notpoxvirus vectors, and thus are not an orthopox, suipox, avipox, orcapripox virus vector. Orthopox include vaccinia, ectromelia, andraccoon pox. One example of an orthopox is vaccinia. Avipox includesfowlpox, canary pox and pigeon pox. Capripox include goatpox andsheeppox. An example of a suipox is swinepox vector. Exemplary pox viralvectors for expression as described for example, in U.S. Pat. No.6,165,460, which is incorporated herein by reference. The vaccinia virusgenome is known in the art. It is composed of a HIND F13L region, TKregion, and an HA region. Recombinant vaccinia virus has been used toincorporate an exogenous gene for expression of the exogenous geneproduct (see, for example, Perkus et al. Science 229:981-984, 1985;Kaufman et al. Inti J. Cancer 48:900-907, 1991; Moss Science 252:1662,1991). Baxby and Paoletti (Vaccine 10:8-9, 1992) disclose theconstruction and use as a vector, of the non-replicating poxvirus,including canarypox virus, fowlpox virus and other avian species. Sutterand Moss (Proc. Nat'l. Acad. Sci U.S.A. 89:10847-10851, 1992) and Sutteret al. (Virology 1994) disclose the construction and use as a vector,the non-replicating recombinant Ankara virus (MVA, modified vacciniaAnkara) in the construction and use of a vector. These vectors are notused in the present methods.

The vectors disclosed herein are also non-yeast vectors. Thus, thedisclosed vectors are not used for expression in yeast such as S.cerevisiae or Kluyveromyces lactis. Thus, the disclosed vectorsgenerally do not include all the required elements for expression inyeast. As examples, promoters are known to be of use in yeast expressionsystems such as the constitutive promoters plasma membrane H⁺-ATPase(PMA1), glyceraldehyde-3-phosphate dehydrogenase (GPD), phosphoglyceratekinase-1 (PGK1), alcohol dehydrogenase-1 (ADH1), and pleiotropicdrug-resistant pump (PDR5). The promoters are not utilized in thepresently disclosed vectors. In addition, many inducible promoters, suchas GAL1-10 (induced by galactose), PHO5 (induced by low extracellularinorganic phosphate), and tandem heat shock HSE elements (induced bytemperature elevation to 37° C.) are not used in the present vectors.Promoters that direct variable expression in response to a titratableinducer include the methionine-responsive MET3 and MET25 promoters andcopper-dependent CUP1 promoters; these promoters are not utilized. Inadditional examples, the vectors do not include yeast nutritionalmarkers (such as URA3, ADE3, HIS1, and others) for selection in yeast.

A number of non-pox non-yeast viral vectors can be utilized, includingpolyoma, SV40 (Madzak et al., 1992, J. Gen. Virol., 73:15331536),adenovirus (Berkner, 1992, Cur. Top. Microbiol. Immunol., 158:39-6;Berliner et al., 1988, Bio Techniques, 6:616-629; Gorziglia et al.,1992, J. Virol., 66:4407-4412; Quantin et al., 1992, Proc. Nad. Acad.Sci. USA, 89:2581-2584; Rosenfeld et al., 1992, Cell, 68:143-155;Wilkinson et al., 1992, Nucl. Acids Res., 20:2233-2239;Stratford-Perricaudet et al., 1990, Hum. Gene Ther., 1:241-256),vaccinia virus (Mackett et al., 1992, Biotechnology, 24:495-499),adeno-associated virus (Muzyczka, 1992, Curr. Top. Microbiol. Immunol.,158:91-123; On et al., 1990, Gene, 89:279-282), herpes viruses includingHSV and EBV (Margolskee, 1992, Curr. Top. Microbiol. Immunol.,158:67-90; Johnson et al., 1992, J. Virol., 66:29522965; Fink et al.,1992. Hum. Gene Ther. 3:11-19; Breakfield et al., 1987, Mol. Neurobiol.,1:337-371; Fresse et al., 1990, Biochem. Pharmacol., 40:2189-2199),Sindbis viruses (H. Herweijer et al., 1995, Human Gene Therapy6:1161-1167; U.S. Pat. Nos. 5,091,309 and 5,2217,879), alphaviruses (S.Schlesinger, 1993, Trends Biotechnol. 11:18-22; I. Frolov et al., 1996,Proc. Natl. Acad. Sci. USA 93:11371-11377), human herpesvirus vectors(HHV) such as HHV-6 and HHV-7, and retroviruses of avian (Brandyopadhyayet al., 1984, Mol. Cell Biol., 4:749-754; Petropouplos et al., 1992, J.Virol., 66:3391-3397), murine (Miller, 1992, Curr. Top. Microbiol.Immunol., 158:1-24; Miller et al., 1985, Mol. Cell Biol., 5:431-437;Sorge et al., 1984, Mol. Cell Biol., 4:1730-1737; Mann et al., 1985, J.Virol., 54:401-407), and human origin (Page et al., 1990, J. Virol.,64:5370-5276; Buchschalcher et al., 1992, J. Virol., 66:2731-2739).Baculovirus (Autographa californica multinuclear polyhedrosis virus;AcMNPV) vectors can be used. Vectors can be obtained from commercialsources (such as PharMingen, San Diego, Calif.; Protein Sciences Corp.,Meriden, Conn.; Stratagene, La Jolla, Calif.). Suitable vectors aredisclosed, for example, in U.S. Published Patent Application No.2010/0247486, which is incorporated herein by reference. In specificnon-limiting examples, the vectors are retrovirus vectors (for example,lentivirus vectors), measles virus vectors, alphavirus vectors,baculovirus vectors, Sindbis virus vectors, adenovirus and poliovirusvectors. These vectors include a polynucleotide that encodes a Brachyuryprotein or Brachyury polypeptide.

Non-pox non-yeast vectors that encode a Brachyury protein or Brachyurypolypeptide include at least one expression control elementoperationally linked to the nucleic acid sequence encoding the Brachyuryprotein or polypeptide. The expression control elements are inserted inthe vector to control and regulate the expression of the nucleic acidsequence. Examples of expression control elements of use in thesevectors includes, but is not limited to, lac system, operator andpromoter regions of phage lambda, promoters derived from polyoma,adenovirus, retrovirus or SV40. Additional operational elements include,but are not limited to, leader sequence, termination codons,polyadenylation signals and any other sequences necessary for theappropriate transcription and subsequent translation of the nucleic acidsequence encoding the Brachyury protein or the Brachyury polypeptide inthe host system. The expression vector can contain additional elementsnecessary for the transfer and subsequent replication of the expressionvector containing the nucleic acid sequence in the host system. Examplesof such elements include, but are not limited to, origins of replicationand selectable markers. It will further be understood by one skilled inthe art that such vectors can be constructed using conventional methods(Ausubel et al., (1987) in “Current Protocols in Molecular Biology,”John Wiley and Sons, New York, N.Y.) and are commercially available.

Optionally, the vector can encode one or more immunostimulatorymolecules, such as IL-2, IL-6, IL-12, LFA (for example, LFA-1, LFA-2and/or LFA-3), CD72, RANTES, G-CSF, GM-CSF, TNF-α, IFN-γ, ICAM-1, B7-1,B7-2, other B7 related molecules, OX-40L or 41 BBL, or combinations ofthese molecules. These immunostimulatory molecules can be used asbiological adjuvants (see, for example, Salgaller et al., 1998, J. Surg.Oncol. 68(2):122-38; Lotze et al., 2000, Cancer J Sci. Am. 6(Suppl1):S61-6, Cao et al., 1998, Stem Cells 16(Suppl 1):251-60; Kuiper etal., 2000, Adv. Exp. Med. Biol. 465:381-90). In several examples, thevector can encode IL-2, RANTES, GM-CSF, TNF-α, IFN-γ, G-CSF, LFA-3,CD72, B7-1, B7-2, B7-1, B7-2, OX-40L, 41 BBL and/or ICAM-1.

Basic techniques for preparing recombinant DNA viruses containing aheterologous DNA sequence encoding a Brachyury protein or Brachyurypolypeptide are known in the art. Such techniques involve, for example,homologous recombination between the DNA sequences flanking the DNAsequence in a donor plasmid and homologous sequences present in aparental virus (Mackett et al., 1982, Proc. Natl. Acad. Sci. USA79:7415-7419). In particular, recombinant viral vectors can be used indelivering the gene. The vector can be constructed, for example, bysteps known in the art, including using a unique restrictionendonuclease site that is naturally present or artificially inserted inthe parental viral vector to insert the heterologous DNA encoding theBrachyury protein or Brachyury polypeptide.

Generally, a DNA donor vector contains the following elements: (i) aprokaryotic origin of replication, so that the vector may be amplifiedin a prokaryotic host; (ii) a gene encoding a marker which allowsselection of prokaryotic host cells that contain the vector (e.g., agene encoding antibiotic resistance); (iii) at least one DNA sequenceencoding the Brachyury protein or Brachyury polypeptide located adjacentto a transcriptional promoter capable of directing the expression of thesequence; and (iv) DNA sequences homologous to the region of the parentvirus genome where the foreign gene(s) will be inserted, flanking theconstruct of element (iii). Methods for constructing donor plasmids forthe introduction of multiple foreign genes into viral virus aredescribed in PCT Publication no WO 91/19803, incorporated herein byreference.

Generally, DNA fragments for construction of the donor vector, includingfragments containing transcriptional promoters and fragments containingsequences homologous to the region of the parent virus genome into whichforeign DNA sequences are to be inserted, can be obtained from genomicDNA or cloned DNA fragments. The donor plasmids can be mono, di-, ormultivalent (i.e., can contain one or more inserted foreign DNAsequences). The donor vector can contain an additional gene that encodesa marker that will allow identification of recombinant virusescontaining inserted foreign DNA. Several types of marker genes can beused to permit the identification and isolation of recombinant viruses.These include genes that encode antibiotic or chemical resistance (e.g.,see Spyropoulos et al., 1988, J. Virol. 62:1046; Falkner and Moss, 1988,J. Virol. 62:1849; Franke et al., 1985, Mol. Cell. Biol. 5:1918), aswell as genes such as the E. coli lacZ gene, that permit identificationof recombinant viral plaques by colorimetric assay (Panicali et al.,1986, Gene 47:193-199).

The DNA gene sequence to be inserted into the virus can be placed into adonor plasmid into which DNA homologous to a section of DNA such as thatof the insertion site of the virus where the DNA is to be inserted hasbeen inserted. Separately the DNA gene sequence to be inserted isligated to a promoter. The promoter-gene linkage is positioned in theplasmid construct so that the promoter-gene linkage is flanked on bothends by DNA homologous to a DNA sequence flanking a region of viral DNAthat is the desired insertion region. With a parental adenoviral vector,an adenoviral promoter is used. Similarly, with a parental lentiviralvector, a lentivirus promoter is used. The resulting plasmid constructis then amplified by growth within E. coli bacteria and isolated. Next,the isolated plasmid containing the DNA gene sequence to be inserted istransfected into a cell culture, along with the parental virus.Recombination between homologous viral DNA in the plasmid and the viralgenome respectively results in a recombinant virus modified by thepresence of the promoter-gene construct in its genome, at a site thatdoes not affect virus viability.

As noted above, the DNA sequence is inserted into a region (insertionregion) in the virus that does not affect virus viability of theresultant recombinant virus. One of skill in the art can readilyidentify such regions in a virus by, for example, randomly testingsegments of virus DNA for regions that allow recombinant formationwithout seriously affecting virus viability of the recombinant.

Homologous recombination between donor plasmid DNA and viral DNA in aninfected cell can result in the formation of recombinant viruses thatincorporate the desired elements. Appropriate host cells for in vivorecombination are generally eukaryotic cells that can be infected by thevirus and transfected by the plasmid vector. Examples of such cellssuitable for use with viral vectors are fibroblasts, HuTK143 (human)cells, and CV-1 and BSC-40 (both monkey kidney) cells. Infection ofcells with a virus and transfection of these cells with plasmid vectorsis accomplished by techniques standard in the art (see U.S. Pat. No.4,603,112 and PCT Publication No. WO 89/03429). Following in vivorecombination, recombinant viral progeny can be identified. For example,co-integration of a gene encoding a marker or indicator gene with theforeign gene(s) of interest, as described above, can be used to identifyrecombinant progeny. One specific non-limiting example of an indicatorgene is the E. coli lacZ gene. Recombinant viruses expressingbeta-galactosidase can be selected using a chromogenic substrate for theenzyme (Panicali et al., 1986, Gene 47:193). Once a recombinant virushas been identified, a variety of well-known methods can be used toassay the expression of the Brachyury protein or Brachyury polypeptideencoded by the inserted DNA fragment. These methods include black plaqueassay (an in situ enzyme immunoassay performed on viral plaques),Western blot analysis, radioimmunoprecipitation (RIPA), and enzymeimmunoassay (EIA).

This disclosure encompasses a recombinant vector comprising more thanone antigen of interest for the purpose of having a multivalent vaccine.For example, the recombinant vectors, such as a viral vector, cancomprise the virus genome or portions thereof, the nucleic acid sequenceencoding the Brachyury protein or Brachyury polypeptide and a nucleicacid sequence encoding a carrier, such as, but not limited to, hepatitisB surface antigen.

The vectors of use in the methods disclosed herein are non-yeast,non-poxviral vectors. Vectors that are useful include adenovirus,alphavirus, lentivirus, measles virus and poliovirus vectors. However,this disclosure is not limited to these types of non-yeast non-poxviralvectors. Additional vectors herpes simplex viruses, human papillomavirus, Simian immunodeficiency viruses, human T cell lymphoma virus(HTLV), human foamy virus, spumaviruses, mammalian type B retroviruses,mammalian type C retroviruses, avian type C retroviruses, mammalian typeD retroviruses. Vectors also include Epstein-Barr virus vectors, Moloneymurine leukemia virus vectors, Harvey murine sarcoma virus vectors,murine mammary tumor virus vectors, Rous sarcoma virus vectors andnonviral plasmid vectors. Several types of vectors of use are disclosedbelow. Compositions including these vectors are of use in inducing aCD4+ T cell response to Brachyury and for the treatment of cancer. Thesecompositions also can be used to induce a CD8+ T cell response toBrachyury.

Adenovirus Vectors

Adenovirus vectors (Ad) vectors can be produced that encode a Brachyuryprotein or a Brachyury polypeptide and are of use in the methodsdisclosed herein. These vectors are of use in the methods disclosedherein, including replication competent, replication deficient, gutlessforms thereof, and adeno-associated virus (AAV) vectors. Without beingbound by theory, adenovirus vectors are known to exhibit strongexpression in vitro, excellent titer, and the ability to transducedividing and non-dividing cells in vivo (Hitt et al., Adv in Virus Res55:479-505, 2000). When used in vivo these vectors lead to strong buttransient gene expression due to immune responses elicited to the vectorbackbone.

Adenoviral vectors are often constructed by insertion of a nucleic acidencoding a Brachyury protein in place of, or in the middle of, essentialviral sequences such as those found at the E1 region of adenovirus(Berkner, BioTechniques, 6:616-629, 1988; Graham et al., Methods inMolecular Biology, 7:109-128, Ed: Murcy, The Human Press Inc., 1991).Inactivation of essential viral genes by, for example, deletion orinsertion, disables the adenovirus' ability to replicate. To propagatesuch vectors in cell culture, the deleted genes must be provided intrans (for example, the E1A and E1B proteins in the case of an E1 deletevector). These replication-defective adenoviruses are produced inpackaging cells engineered to complement the replication-incompetentvirus by expressing the subset of genetic elements deleted from theirviral genome. Potential sites for the insertion of a nucleic acid ofinterest, such as a nucleic acid encoding a Brachyury protein, inrecombinant adenoviral vectors include, without limitation, the E1, E2,E3 and the E4 region. In some embodiments, a recombinant adenoviralvector is produced from a human adenovirus that has the E1 regiondeleted and replaced with a nucleic acid encoding a Brachyury protein orBrachyury polypeptide. The resulting viral vector, with one or more ofits essential genes inactivated, is replication defective(Statford-Perricaudet et al., Human Gene Therapy, 1:241-256, 1990).

The recombinant adenovirus vectors can include: (1) a packaging siteenabling the vector to be incorporated into replication-defective Advirions; and (2) the nucleic acid encoding the Brachyury protein orBrachyury polypeptide. Other elements of use for incorporation intoinfectious virions, include the 5′ and 3′ Ad ITRs; the E2 and E3 genescan be included in the vector. In some embodiments, a nucleic acidencoding a Brachyury protein or Brachyury polypeptide is inserted intoadenovirus in the deleted E1A, E1B or E3 region of the virus genome. Insome embodiments, the adenovirus vectors do not express one or morewild-type adenovirus gene products, such as E1a, E1b, E2, E3, E4. Insome non-limiting examples, virions are typically used together withpackaging cell lines that complement the functions of E1, E2A, E4 andoptionally the E3 gene regions (see, for example, U.S. Pat. Nos.5,872,005, 5,994,106, 6,133,028 and 6,127,175, incorporated by referenceherein in their entirety). Adenovirus vectors can be purified andformulated using techniques known in the art.

In some embodiments, packaging cell lines such as the human embryonickidney 293 (“HEK-293” or “293”) cell line (Graham et al., J. Gen.Virol., 36:59-72, 1977) or human embryonic retinoblast (“HER-911” or“911”) cell line (Fallaux et al., Hum. Gene Ther., 7:215-222, 1996),provide in trans the missing region, such as the E1 region, so that thedeleted or modified adenoviral vector can replicate in such cells.Suitable adenoviral vectors are disclosed, for example, in U.S. PatentPublication No. 20080193484, which is incorporated herein by reference.Replication-defective adenovirus virions encapsulating the recombinantadenovirus vectors can be made by standard techniques known in the artusing packaging cells and packaging technology. Examples of thesemethods can be found, for example, in U.S. Pat. No. 5,872,005,incorporated herein by reference in its entirety.

Recombinant AAV vectors are characterized in that they are capable ofdirecting the expression and the production of the selected transgenicproducts in targeted cells. Thus, the recombinant vectors comprise atleast all of the sequences of AAV essential for encapsidation and thephysical structures for infection of target cells.

Recombinant AAV (rAAV) virions can be constructed such that theyinclude, as operatively linked components in the direction oftranscription, control sequences including transcriptional initiationand termination sequences, and the nucleic acid encoding the Brachyuryprotein or Brachyury polypeptide. These components are bounded on the 5′and 3′ end by functional AAV inverted terminal repeat (ITR) sequences.By “functional AAV ITR sequences” is meant that the ITR sequencesfunction as intended for the rescue, replication and packaging of theAAV virion. Hence, AAV ITRs for use in the vectors need not have awild-type nucleotide sequence, and can be altered by the insertion,deletion or substitution of nucleotides, or the AAV ITRs can be derivedfrom any of several AAV serotypes, provided they are functional. An AAVvector is a vector derived from an adeno-associated virus serotype,including without limitation, AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6,AAV-7, AAV-8, etc. In some embodiments, the AAV vectors have the wildtype REP and CAP genes deleted in whole or part, but retain functionalflanking ITR sequences. These vectors can all be used, withoutlimitation, for the expression of a Brachyury protein.

Alphavirus

Alphaviruses encoding a Brachyury protein or Brachyury polypeptide areprovided and are of use in the methods disclosed herein. Alphavirusesare a set of serologically related arthropod-borne viruses of theTogavirus family. Twenty-six known viruses and virus subtypes have beenclassified within the alphavirus genus utilizing the hemagglutinationinhibition (HI) assay. Briefly, the HI test segregates the 26alphaviruses into three major complexes: the Venezuelan encephalitis(VE) complex, the Semliki Forest (SF) complex, and the westernencephalitis (WE) complex. In addition, four additional viruses, easternencephalitis (EE), Barmah Forest, Middelburg, and Ndumu, receiveindividual classification based on the HI serological assay.Representative examples of suitable alphaviruses include Aura (AmericanType Culture Collection (ATCC) VR-368), Bebaru virus (ATCC VR-600, ATCCVR-1240), Cabassou (ATCC VR-922), Chikungunya virus (ATCC VR-64, ATCCVR-1241), Eastern equine encephalomyelitis virus (ATCC VR-65, ATCCVR-1242), Fort Morgan (ATCC VR-924), Getah virus (ATCC VR-369, ATCCVR-1243), Kyzylagach (ATCC VR-927), Mayaro (ATCC VR-66), Mayaro virus(ATCC VR-1277), Middleburg (ATCC VR-370), Mucambo virus (ATCC VR-580,ATCC VR-1244), Ndumu (ATCC VR-371), Pixuna virus (ATCC VR-372, ATCCVR-1245), Ross River virus (ATCC VR-373, ATCC VR-1246), Semliki Forest(ATCC VR-67, ATCC VR-1247), Sindbis virus (ATCC VR-68, ATCC VR-1248),Tonate (ATCC VR-925), Triniti (ATCC VR-469), Una (ATCC VR-374),Venezuelan equine encephalomyelitis (ATCC VR-69), Venezuelan equineencephalomyelitis virus (ATCC VR-923, ATCC VR-1250 ATCC VR-1249, ATCCVR-532), Western equine encephalomyelitis (ATCC VR-70, ATCC VR-1251,ATCC VR-622, ATCC VR-1252), Whataroa (ATCC VR-926), and Y-62-33 (ATCCVR-375), see U.S. Pat. No. 5,843,723, which is incorporated herein byreference.

In some embodiments, and alphavirus vector is a Sinbis virus. In someembodiments, recombinant alphavirus vector constructs are utilized thatinclude a 5′ sequence which is capable of initiating transcription of analphavirus, a nucleotide sequence encoding alphavirus nonstructuralproteins, a viral junction region which has been inactivated such thatviral transcription of the subgenomic fragment is prevented, analphavirus RNA polymerase recognition sequence, and a nucleic acidsequence encoding a Brachyury protein. Alphavirus vector constructswhich have inactivated viral junction regions do not transcribe thesubgenomic fragment, making them suitable for a wide variety ofapplications.

In some embodiments, the alphavirus such as Sinbis virus, constructs areprovided which contain a 5′ promoter which is capable of initiating thesynthesis of viral RNA in vitro from cDNA. The 5′ promoters include botheukaryotic and prokaryotic promoters, such as, for example, theβ-galactosidase promoter, trpE promoter, lacZ promoter, T7 promoter, T3promoter, SP6 promoter, SV40 promoter, CMV promoter, and MoMLV LTR.Representative examples of such sequences include nucleotides 1-60, andto a lesser extent nucleotides 150-210, of the wild-type Sindbis virus,nucleotides 10-75 for tRNA Asparagine (Schlesinger et al., U.S. Pat. No.5,091,309, incorporated herein by reference), and 5′ sequences fromother Togaviruses which initiate transcription.

Alphavirus vectors can contain sequences which encode alphavirusnonstructural proteins (NSPs). As an example, for Sindbis virus thereare four nonstructural proteins, NSP1, NSP2, NSP3 and NSP4, which encodeproteins that enable the virus to self-replicate. Nonstructural proteins1 through 3 (NSP1-NSP3) are, encoded by nucleotides 60 to 5750 of thewild-type Sindbis virus. These proteins are produced as a polyproteinand later cleaved into nonstructural proteins NSP1, NSP2, and NSP3.NSP4. The alphavirus vector constructs can also include a viral junctionregion which has been inactivated, such that viral transcription of thesubgenomic fragment is prevented. Briefly, the alphavirus viral junctionregion normally controls transcription initiation of the subgenomicmRNA. In the case of the Sindbis virus, the normal viral junction regiontypically begins at approximately nucleotide number 7579 and continuesthrough at least nucleotide number 7612 (and possibly beyond), see U.S.Pat. No. 5,843,723 for the complete sequence, incorporated herein byreference.

Several members of the alphavirus genus can be used as “replicon”expression vectors. Replicon vectors may be utilized in any of severalformats, including DNA vector constructs, RNA replicon vectors, andrecombinant replicon particles (see below). These include, for example,SIN (Xiong et al., Science 243:1188-1191, 1989; Dubensky et al., J.Virol. 70:508-519, 1996; Hariharan et al., J. Virol. 72:950-958, 1998;Polo et al., PNAS 96:4598-4603, 1999), Semliki Forest virus (Liljestrom,Bio/Technology 9:1356-1361, 1991; Berglund et al., Nat. Biotech.16:562-565, 1998), VEE (Pushko et al. Virology 239:389-401, 1997), andchimeras of multiple alphaviruses (U.S. Pat. No. 6,376,236; PCTPublication No. WO2002099035; Perri et al., J. Virol. 77:10394-10403,2003).

Alphavirus vector constructs are also disclosed in U.S. Pat. Nos.5,789,245; 5,843,723; 5,814,482, and 6,015,694; PCT Publication No. WO00/61772; and PCT Publication No. WO 02/99035. Generally, these vectorsinclude a 5′ sequence which initiates transcription of alphavirus RNA, anucleotide sequence encoding alphavirus nonstructural proteins, a viralsubgenomic junction region promoter which directs the expression of anadjacent heterologous nucleic acid sequence, an RNA polymeraserecognition sequence and a polyadenylate tract.

An alphavirus can be used as a replicon (a recombinant alphavirusparticle) that is a virus-like particle containing a self-replicatingalphavirus vector or “replicon” nucleic acid. The replicon particleitself is generally considered to be replication incompetent or“defective,” that is no progeny replicon particles will result when ahost cell is infected with a replicon particle, because genes encodingone or more structural proteins necessary for packaging are deleted.

Although alphavirus vectors can be used directly for administration invivo as RNA, or delivered as a plasmid-based cDNA (e.g., EukaryoticLayered Vector Initiation System), often, for in vivo vaccine andtherapeutic applications, the alphavirus RNA replicon vector or repliconRNA is first packaged into a virus-like particle, comprising alphavirusstructural proteins (e.g., capsid protein and envelope glycoproteins).Alphavirus and replicons of use are disclosed, for example, in PublishedU.S. Patent Application No. 20110002958, which is incorporated herein byreference. Because of their configuration, vector replicons do notexpress these alphavirus structural proteins necessary for packaginginto recombinant alphavirus replicon particles. Thus, to generatereplicon particles, the structural proteins must be provided in trans.

Packaging can be accomplished by a variety of methods, includingtransient approaches such as co-transfection of in vitro transcribedreplicon and defective helper RNA(s) (Liljestrom, Bio/Technology9:1356-1361, 1991; Bredenbeek et al., I Virol. 67:6439-6446, 1993;Frolov et al., J. Virol. 71:2819-2829, 1997; Pushko et al., Virology239:389-401, 1997; U.S. Pat. Nos. 5,789,245 and 5,842,723) or plasmidDNA-based replicon and defective helper constructs (Dubensky et al., J.Virol. 70:508-519, 1996), as well as introduction of alphavirusreplicons into stable packaging cell lines (PCL) (Polo et al., PNAS96:4598-4603, 1999; U.S. Pat. Nos. 5,789,245; 5,842,723; 6,015,694).

The trans packaging methodologies permit the modification of one or morestructural protein genes (for example, to incorporate sequences ofalphavirus variants such as the attenuated mutants, see U.S. Pat. Nos.5,789,245; 5,842,723; 6,015,694), followed by the subsequentincorporation of the modified structural protein into the final repliconparticles. In addition, such packaging permits the overall modificationof alphavirus replicon particles by packaging of a vector construct orRNA replicon derived from a first alphavirus using structural proteinsderived from a second alphavirus different from that of the vectorconstruct.

Measles Virus

Measles viruses encoding a Brachyury protein or Brachyury polypeptideare provided and are of use in the methods disclosed herein. The nucleicacid sequences of Measles Viruses are disclosed in PCT Publication No.WO 98/13501, which provides the sequence of a DNA copy of the positivestrand (antigenomic) message sense RNA of various wild-type of vaccinemeasles strains, including Edmonston Wild-type strain, Moraten strainand Schwarz strain. PCT Publication No. WO 97/06270, incorporated hereinby reference, discloses the production of recombinant measles vectors.

An attenuated strain of measles virus can also be used to deliver aBrachyury protein or Brachyury polypeptide. The Moraten attenuated formof the virus has been used world-wide as a vaccine and has an excellentsafety record (Hilleman, et al., J. Am. Med. Assoc. 206: 587-590, 1968).Accordingly, in one embodiment, the Moraten strain is used. The Moratenvaccine is commercially available from MERCK® and is providedlyophilized in a vial which when reconstituted to 0.5 ml comprises 10³pfu/ml.

In a further embodiment, the Edmonston-B vaccine strain of measles virusis used (MV-Edm) (Enders and Peebles, Proc. Soc. Exp. Biol. Med. 86:277-286, 1954). MV-Edm grows efficiently in tumor cells but its growthis severely restricted in primary cultures of human peripheral bloodmononuclear cells, normal dermal fibroblasts, and vascular smooth musclecells. A form of the Enders attenuated Edmonston strain is availablecommercially from Merck (ATTENUVAX®). Other attenuated measles virusstrains can also be utilized, such as Leningrad-16, and Moscow-5 strains(Sinitsyna, et al., Res. Virol. 141(5): 517-31, 1990), Schwarz strain(Fourrier, et al., Pediatrie 24(1): 97-8, 1969), 9301B strain (Takeda,et al. J. VIROL. 72/11: 8690-8696), the AIK-C strain (Takehara, et al.,Virus Res 26 (2): 167-75, 1992), and those described inSchneider-Shaulies, et al., PNAS 92(2): 3943-7, 1995).

In some embodiments, the recombinant measles virus nucleotide sequencecomprises a replicon having a total number of nucleotides which is amultiple of six. The “rule of six” is expressed in the fact that thetotal number of nucleotides present in the recombinant cDNA finallyamount to a total number of nucleotides which is a multiple of six, arule which allows efficient replication of genome RNA of the measlesvirus.

In additional embodiments, heterologous DNA, such as a nucleic acidencoding Brachyury protein, is cloned in the measles virus within anAdditional Transcription Unit (ATU) inserted in the cDNA correspondingto the antigenomic RNA of measles virus. The location of the ATU canvary along the cDNA: it is however located in such a site that it willbenefit from the expression gradient of the measles virus. Therefore,the ATU can be spread along the cDNA. In one embodiment, the ATU isinserted in the N-terminal portion of the sequence and especially withinthe region upstream from the L-gene of the measles virus and upstreamfrom the M gene of the virus. In other embodiments, the ATU is insertedupstream from the N gene of the virus, see U.S. Published PatentApplication No. 2011/0129493, incorporated herein by reference.Particular cistrons in the measles virus genome can targeted to modifygenes whose expression is associated with attenuation(Schneider-Shaulies et at. PNAS 92(2): 3943-7, 1995, Takeda, et al. J.Virol. 72/11: 8690-8696, 1998). Thus, in one embodiment, a recombinantmeasles virus strain is generated encoding a Brachyury protein orBrachyury polypeptide in any of an H protein, a V protein, a C protein,and combinations thereof.

Recombinant measles virus vectors include the plasmid pTM-MVSchw whichcontains the cDNA resulting from reverse transcription of theantigenomic RNA of measles virus and an adapted expression controlsequence including a promoter and terminator for the T7 polymerase.Vectors are also disclosed, for example, in U.S. Published PatentApplication No. 2006/0013826, which is incorporated herein by reference.These vectors are of use in the methods disclosed herein.

Additional attenuated strains of measles virus can be produced thatexpress a Brachyury protein or Brachyury polypeptide. Attenuated strainsof viruses are obtained by serial passage of the virus in cell culture(e.g., in non-human cells), until a virus is identified which isimmunogenic but not pathogenic. While wild type virus will cause fatalinfection in marmosets, vaccine strains do not. Individuals receiving anattenuated measles virus vaccine do not display classical measlessymptoms. Attenuation is associated with decreased viral replication (asmeasured in vivo by inability to cause measles in monkeys), diminishedviremia, and failure to induce cytopathological effects in tissues(e.g., cell-cell fusion, multinucleated cells). See U.S. Pat. No.7,393,527, which is incorporated herein by reference.

In one embodiment, an effective dose of an attenuated measles virusencoding a Brachyury protein or Brachyury polypeptide is produced byinfecting a primary cell or a continuous cell line with a startinginnoculum of a stock comprising an attenuated Moraten strain of measlesvirus (or an innoculum of an MMR stock) or the MV-Edm strain or any ofthe other strains described above and expanding the virus after serialpassage. Cells or cell lines include, but are not limited to, monkeykidney or testes cells or monkey cell lines (e.g., Vero, KB, CV-1,BSC-1, and the like). Viral replication in cells is observed ascell-cell fusion and syncytia formation.

The attenuated measles virus is expanded until a desired doseconcentration is obtained in standard cell culture media. In oneembodiment, the therapeutically effective dose concentration is about10³ to 10¹² pfu. In another embodiment of the invention, theconcentration is about 10⁵ to 10⁸ pfu. Viral titer can be assayed byinoculating cells (e.g., Vero cells) in culture dishes (e.g., such as 35mm dishes). After 2-3 hours of viral adsorption, the inoculum is removedand cells are overlaid with a mixture of cell culture medium and agaroseor methylcellulose (e.g., 2 ml DMEM containing 5% FCS and 1% SeaPlaqueagarose). After about 3 to about 5 days, cultures are fixed with 1 ml of10% trifluoroacetic acid for about 1 hour, then UV cross-linked for 30minutes. After removal of the agarose overlay, cell monolayers arestained with crystal violet and plaques are counted to determine viraltiter. Virus is harvested from cell syncytia by scraping cells from thedishes, subjecting them to freeze/thawing (e.g., approximately tworounds), and centrifuging. The cleared supernatants represent “plaquepurified” virus.

Viral stocks are produced by infection of cell monolayers (e.g.,adsorption for about 1.5 hours at 37° C.), followed by scraping ofinfected cells into a suitable medium (e.g., Opti-MEM, Gibco-BRL) andfreeze/thaw lysis (for example, 2 rounds). Viral stocks are aliquoted,frozen and stored at 70° C.-80° C. and can be stored at concentrationshigher than the therapeutically effective dose. In one embodiment, viralstock is stored in a stabilizing solution. Stabilizing solutions areknown in the art, see for example, U.S. Pat. Nos. 4,985,244, and4,500,512.

Poliovirus

Polioviruses encoding a Brachyury protein or a Brachyury polypeptide areprovided and are of use in the methods disclosed herein. The entirepoliovirus genome has been cloned and sequenced and the viral proteinsidentified. An infectious poliovirus cDNA is also available which hasallowed further genetic manipulation of the virus (Racaniello V R etal., Science 214(4542) 916-919, 1981). The wild-type genomic RNAmolecule is 7433 nucleotides long, polyadenylated at the 3′ end and hasa small covalently attached viral protein (VPg) at the 5′ terminus(Kitamura N et al., Nature 291:547-553; 1981 Racaniello V R et al.,Proc. Natl. Acad. Sci. USA 78:4887-4891, 1981). Expression of thepoliovirus genome occurs via the translation of a single protein(polyprotein) which is subsequently processed by virus encoded proteases(2A and 3C) to give the mature structural (capsid) and nonstructuralproteins (Kitamura N et al., Nature 291:547-553, 1981; Koch F et al.,The Molecular Biology of Poliovirus, Springer-Verlag, Vienna, 1985).Poliovirus replication is catalyzed by the virus-encoded RNA-dependentRNA polymerase, which copies the genomic RNA to give a complementary RNAmolecule, which then serves as a template for further RNA production(Koch F et al., supra; Kuhn R J et al., in D J Rowlands et al. (ed.)Molecular Biology of Positive Strand RNA viruses, Academic Press Ltd.,London, 1987). The translation and proteolytic processing of thepoliovirus polyprotein is described in Nicklin M J H et al.,Bio/Technology 4:33-42, 1986.

The viral RNA genome encodes the necessary proteins required forgeneration of new progeny RNA, as well as encapsidation of the new RNAgenomes. In vitro, poliovirus is lytic, resulting in the completedestruction of permissive cells. Since the viral replication cycle doesnot include any DNA intermediates, there is no possibility ofintegration of viral DNA into the host chromosomal DNA.

Early studies identified three poliovirus types based on reactivity toantibodies (Koch F et al., supra, 1985). These three serological types,designated as type I, type II, and type III, have been furtherdistinguished as having numerous nucleotide differences in both thenon-coding regions and the protein coding regions. All three strains aresuitable for use in delivering heterologous proteins. In addition, thereare also available attenuated versions of all three strains ofpoliovirus.

Replicons can comprise deoxyribonucleic acid (DNA) or ribonucleic acid(RNA). Replicons are poliovirus-based polynucleotides that lack a wildtype poliovirus nucleic acid necessary for encapsidation of the virus.Consequently, newly encapsidated replicons cannot be produced followinginitial cell entry in the absence of the missing nucleic acid. Repliconscan lack this nucleic acid as a result of any modification of thewildtype poliovirus nucleic acid including, but not limited to,deletions, insertions, and substitutions, including an insertion of anucleic acid encoding a Brachyury protein. In some embodiments,poliovirus replicons lack a wild type poliovirus nucleic acid thatencodes at least a portion of a protein that is required forencapsidation. Proteins necessary for replicon encapsidation includeproteins that are part of the capsid structure. Examples of suchproteins are those encoded by the VP1, VP2, VP3, and VP4 genes of thepoliovirus P1 capsid precursor region, the Vpg protein, and thoseproteins that are necessary for proper processing of structural proteinsof the capsid structure, such as the proteases responsible for cleavingthe viral polyprotein. Thus, in some embodiments, the poliovirus vectorlacks nucleic acid sequences encoding one or more of VP1, VP2, VP3, andVP4, genes of the poliovirus P1 capsid precursor region, the Vpgprotein, and encodes a Brachyury protein or Brachyury polypeptide.

Replicons are typically introduced into a cell in an RNA form.Encapsidated replicons are able to enter cells via interaction of thecapsid proteins with poliovirus receptor. Replicons are fully capable ofRNA replication (amplification) upon introduction into cells andtranslation, in the correct reading frame, of the single polyproteinthrough which expression of the entire replicon genome occurs.Translation of replicon sequences may be transient, usually lasting onlyabout 24-48 hours. High levels of replicon-encoded proteins canaccumulate during the translation period. Encapsidated replicons areable to enter cells via interaction of the capsid proteins with the hPVRprotein.

In some embodiments, replicons comprise RNA, including sequencesencoding Brachyury protein, and are encapsidated. In some examples, thereplicons have a deletion of the capsid (P1) gene and are derived fromthe RNA genome of poliovirus type 1, type 2, type 3 or combinationsthereof. Further, a nucleic acid encoding a Brachyury protein orBrachyury polypeptide can be substituted for part or all of the capsid(P1) gene such that the portion of the capsid (P1) gene which remains,if any, is insufficient to support encapsidation in vivo. Generally, theterm “P1 replicons” refers to replicons in which the entire nucleic acidencoding the P1 capsid precursor protein has been deleted or alteredsuch that the proteins which are normally encoded by this nucleic acidare not expressed or are expressed in a non-functional form. Theproteins that are normally encoded by the P1 capsid precursor region ofthe poliovirus genome include the proteins encoded by the VP1, VP2, VP3,and VP4 genes. P1 replicons, therefore, lack the VP1, VP2, VP3, and VP4genes or comprise unexpressible or non-functional forms of the VP1, VP2,VP3, and VP4 genes. P1 replicons can include a nucleic acid encoding aBrachyury protein or Brachyury polypeptide substituted for the VP1, VP2,VP3, and VP4 genes.

In some embodiments, encapsidated replicons may be produced byintroducing both a replicon and a complementing expression vector thatprovides the missing nucleic acid necessary for encapsidation in transto a host cell. A “replicon encapsidation vector” refers to anon-poliovirus-based vector that comprises a nucleic acid required forreplicon encapsidation and provides the required nucleic acid (orencoded protein) in trans. Replicon encapsidation vectors can beintroduced into a host cell prior to, concurrently with, or subsequentto replicon introduction. Suitable methods for encapsidation aredisclosed in U.S. Pat. No. 6,680,169, which is incorporated by referenceherein. Methods which can be used to prepare encapsidated replicons havebeen described Porter D C et al., J. Virol. 67:3712-3719, 1993; Porter DC et al., 1995, J. Virol. 69:1548-1555, 1995; PCT Publication No. WO96/25173; U.S. Pat. Nos. 5,614,413, 5,817,512; 6,063,384; and 6,680,169.

Nonencapsidated replicons can be delivered directly to target cells, forexample by direct injection into, for example, muscle cells (see, forexample, Acsadi G et al., Nature 352(6338):815-818, 1991; Wolff J A etal., Science 247:1465-1468, 1990), or by electroporation, transfectionmediated by calcium phosphate, transfection mediated by DEAE-dextran,liposome-mediated transfection or receptor-mediated nucleic acid uptake(see for example Wu G et al., J. Biol. Chem. 263:14621-14624, 1988;Wilson J M et al., J. Biol. Chem. 267:963-967, 1992; and U.S. Pat. No.5,166,320), or other methods of delivering naked nucleic acids to targetcells.

Retroviral Vectors

Retroviral vectors, including lentiviral vectors encoding a Brachyuryprotein and/or Brachyury polypeptide are provided and are of use in themethods disclosed herein. Retroviral vectors have been tested and foundto be suitable delivery vehicles for the stable introduction of avariety of genes of interest into the genomic DNA of a broad range oftarget cells. Without being bound by theory, the ability of retroviralvectors to deliver unrearranged, single copy transgenes into cells makesretroviral vectors well suited for transferring genes into cells.Further, retroviruses enter host cells by the binding of retroviralenvelope glycoproteins to specific cell surface receptors on the hostcells. Consequently, pseudotyped retroviral vectors in which the encodednative envelope protein is replaced by a heterologous envelope proteinthat has a different cellular specificity than the native envelopeprotein (e.g., binds to a different cell-surface receptor as compared tothe native envelope protein) can also be used.

Generally, retroviruses contain three major coding domains, gag, pol,env, which code for essential virion proteins. Retroviral vectors are ofuse wherein gag, pol and/or env are absent or not functional. Retroviralvectors are disclosed, for example, in U.S. Published Patent ApplicationNo. 20060286634, which is incorporated herein by reference herein.

Thus retroviral vectors are provided which include, for example,retroviral transfer vectors comprising a nucleic acid encoding aBrachyury protein and retroviral packaging vectors comprising one ormore packaging elements. In some embodiments, pseudotyped retroviralvectors are provided encoding a heterologous or functionally modifiedenvelope protein for producing pseudotyped retrovirus.

There are many retroviruses and examples include: murine leukemia virus(MLV), lentivirus such as human immunodeficiency virus (HIV), equineinfectious anaemia virus (EIAV), mouse mammary tumor virus (MMTV), Roussarcoma virus (RSV), Fujinami sarcoma virus (FuSV), Moloney murineleukemia virus (Mo-MLV), FBR murine osteosarcoma virus (FBR MSV),Moloney murine sarcoma virus (Mo-MSV), Abelson murine leukemia virus(A-MLV), Avian myelocytomatosis virus-29 (MC29), and Avianerythroblastosis virus (AEV). Other retroviruses suitable for useinclude, but are not limited to, Avian Leukosis Virus, Bovine LeukemiaVirus, Mink-Cell Focus-Inducing Virus. The core sequence of theretroviral vectors can be derived from a wide variety of retroviruses,including for example, B, C, and D type retroviruses as well asspumaviruses and lentiviruses (see RNA Tumor Viruses, Second Edition,Cold Spring Harbor Laboratory, 1985). An example of a retrovirussuitable for use in the compositions and methods disclosed herein,includes, but is not limited to, lentivirus.

One lentivirus is a human immunodeficiency virus (HIV), for example,type 1 or 2 (i.e., HIV-1 or HIV-2). Other lentivirus vectors includesheep Visna/maedi virus, feline immunodeficiency virus (FIV), bovinelentivirus, simian immunodeficiency virus (SIV), an equine infectiousanemia virus (EIAV), and a caprine arthritis-encephalitis virus (CAEV).

Lentiviruses share several structural virion proteins in common,including the envelope glycoproteins SU (gp120) and TM (gp41), which areencoded by the env gene; CA (p24), MA (p117) and NC (p7-11), which areencoded by the gag gene; and RT, PR and IN encoded by the pol gene.HIV-1 and HIV-2 contain accessory and other proteins involved inregulation of synthesis and processing virus RNA and other replicativefunctions. The accessory proteins, encoded by the vif, vpr, vpu/vpx, andnef genes, can be omitted (or inactivated) from the recombinant system.In addition, tat and rev can be omitted or inactivated, such as bymutation or deletion.

Without being bound by theory, the use of lentivirus-based gene transfertechniques generally relies on the in vitro production of recombinantlentiviral particles carrying a highly deleted viral genome in which agene of interest, such as a nucleic acid encoding a Brachyury protein orBrachyury polypeptide, is accommodated. In particular, the recombinantlentivirus are recovered through the in trans co-expression in apermissive cell line of (1) the packaging constructs, i.e., a vectorexpressing the Gag-Pol precursors together with Rev (alternativelyexpressed in trans); (2) a vector expressing an envelope receptor,generally of an heterologous nature, and (3) the transfer vector,consisting in the viral cDNA deprived of all open reading frames, butmaintaining the sequences required for replication, incapsidation, andexpression, in which the sequences to be expressed are inserted. In oneembodiment the lentigen lentiviral vector described in Lu, X. et al.Journal of gene medicine 6:963-973, 2004 is used to express theBrachyury protein or Brachyury polypeptide. Suitable lentiviral vectorsare also disclosed, for example, in U.S. Published Patent ApplicationNo. 20100062524, which is incorporated herein by reference.

Retroviral packaging systems for generating producer cells and producercell lines that produce retroviruses, and methods of making suchpackaging systems are known in the art. Generally, the retroviralpackaging systems include at least two packaging vectors: a firstpackaging vector which includes a first nucleotide sequence comprising agag, a pol, or gag and pol genes; and a second packaging vector whichincludes a second nucleotide sequence comprising a heterologous orfunctionally modified envelope gene. In some embodiments, the retroviralelements are derived from a lentivirus, such as HIV. These vectors canlack a functional tat gene and/or functional accessory genes (vif, vpr,vpu, vpx, nef). In other embodiments, the system further comprises athird packaging vector that comprises a nucleotide sequence comprising arev gene. The packaging system can be provided in the form of apackaging cell that contains the first, second, and, optionally, thirdnucleotide sequences.

First generation lentiviral vector packaging systems provide separatepackaging constructs for gag/pol and env, and typically employ aheterologous or functionally modified envelope protein for safetyreasons. In second generation lentiviral vector systems, the accessorygenes, vif, vpr, vpu and nef, are deleted or inactivated. Thirdgeneration lentiviral vector systems are those from which the tat genehas been deleted or otherwise inactivated (e.g., via mutation).Compensation for the regulation of transcription normally provided bytat can be provided by the use of a strong constitutive promoter, suchas the human cytomegalovirus immediate early (HCMV-IE)enhancer/promoter. Other promoters/enhancers can be selected based onstrength of constitutive promoter activity, specificity for targettissue (e.g., liver-specific promoter), or other factors relating todesired control over expression, as is understood in the art. Forexample, in some embodiments, an inducible promoter such as tet can beused to achieve controlled expression. The gene encoding rev can beprovided on a separate expression construct, such that a typical thirdgeneration lentiviral vector system will involve four plasmids: one eachfor gagpol, rev, envelope and the transfer vector. Regardless of thegeneration of packaging system employed, gag and pol can be provided ona single construct or on separate constructs.

Typically, the packaging vectors are included in a packaging cell, andare introduced into the cell via transfection, transduction orinfection. Methods for transfection, transduction or infection are wellknown by those of skill in the art. A retroviral vector of the presentinvention can be introduced into a packaging cell line, viatransfection, transduction or infection, to generate a producer cell orcell line. The packaging vectors can be introduced into human cells orcell lines by standard methods including, for example, calcium phosphatetransfection, lipofection or electroporation. In some embodiments, thepackaging vectors are introduced into the cells together with a dominantselectable marker, such as neo, DHFR, Gin synthetase or ADA, followed byselection in the presence of the appropriate drug and isolation ofclones. A selectable marker gene can be linked physically to genesencoding by the packaging vector.

Stable cell lines, wherein the packaging functions are configured to beexpressed by a suitable packaging cell, are known. For example, see U.S.Pat. No. 5,686,279; and Ory et al., Proc. Natl. Acad. Sci.93:11400-11406, 1996, which describe packaging cells. Zufferey et al.,Nature Biotechnology 15:871-875, 1997 disclose a lentiviral packagingplasmid wherein sequences 3′ of pol including the HIV-1 envelope geneare deleted. The construct contains tat and rev sequences and the 3′ LTRis replaced with poly A sequences. The 5′ LTR and psi sequences arereplaced by another promoter, such as one which is inducible. Forexample, a CMV promoter can be used.

The packaging vectors can include additional changes to the packagingfunctions to enhance lentiviral protein expression and to enhancesafety. For example, all of the HIV sequences upstream of gag can beremoved. Also, sequences downstream of envelope can be removed.Moreover, steps can be taken to modify the vector to enhance thesplicing and translation of the RNA.

A self-inactivating vector (SIN) can be used, which improves thebiosafety of the vector by deletion of the HIV-1 long terminal repeat(LTR) as described, for example, by Zufferey et al., J. Virology72(12):9873-9880, 1998. Inducible vectors can also be used, such asthrough a tet-inducible LTR.

Host Cells

DNA sequences encoding a Brachyury protein and/or Brachyury polypeptidecan be expressed from a vector in vitro by DNA transfer into a suitablehost cell. The term “host cell” also includes any progeny of the subjecthost cell. It is understood that all progeny may not be identical to theparental cell since there may be mutations that occur duringreplication. Methods of stable transfer, meaning that the foreign DNA iscontinuously maintained in the host, are known in the art.

Hosts cells can include microbial, insect and mammalian host cells.Methods of expressing DNA sequences having eukaryotic or viral sequencesin prokaryotes are well known in the art. Non-limiting examples ofsuitable host cells include animal cells (for example, mammalian cells,such as human). Techniques for the propagation of mammalian cells inculture are well-known (see, Jakoby and Pastan (eds), 1979, CellCulture. Methods in Enzymology, volume 58, Academic Press, Inc.,Harcourt Brace Jovanovich, N.Y.). Examples of commonly used mammalianhost cell lines are VERO and HeLa cells, CHO cells, and WI38, BHK, andCOS cell lines, although cell lines may be used, such as cells designedto provide higher expression desirable glycosylation patterns, or otherfeatures.

Transformation of a host cell with recombinant DNA can be carried out byconventional techniques as are well known to those skilled in the art.Where the host is prokaryotic, such as, but not limited to, E. coli,competent cells which are capable of DNA uptake can be prepared fromcells harvested after exponential growth phase and subsequently treatedby the CaCl₂ method using procedures well known in the art.Alternatively, MgCl₂ or RbCl can be used. Transformation can also beperformed after forming a protoplast of the host cell if desired, or byelectroporation.

When the host is a eukaryote, such methods of transfection of DNA ascalcium phosphate coprecipitates, conventional mechanical proceduressuch as microinjection, electroporation, insertion of a plasmid encasedin liposomes, or infection with the poxvirus vectors can be used.Eukaryotic cells can also be co-transformed with polynucleotidesequences encoding a Brachyury protein or Brachyury polypeptide, and asecond foreign DNA molecule encoding a selectable phenotype, such as theherpes simplex thymidine kinase gene. Methods for using viral vectors totransform eukaryotic cells are known, (see for example, Eukaryotic ViralVectors, Cold Spring Harbor Laboratory, Gluzman ed., 1982).

A nucleic acid encoding a Brachyury protein is expressed in the hostcell, such as a Salmonella host cell, for example S. typhimurium hostcell, or a Listeria, such as a L. monocytogenes host cell. A hostcell-compatible promoter is any promoter or promoter/enhancer that isable to initiate sufficient transcription of the Brachyury protein orBrachyury polypeptide, such as in amounts sufficient to induce aBrachyury specific CD4+ T cell response when the host cell is introducedinto the subject. Some examples of expression control sequences are apromoter/enhancer from the cytomegalovirus (CMV) immediate early gene 1or the Rous sarcoma virus (RSV) long terminal repeat or the simian virus40 promoter or the adenovirus 2 major late promoter or the mouse mammarytumor virus promoter (MMTV). These host cells can be used in attenuatedand/or heat-killed forms. These host cells can be administered multipletimes without eliciting host neutralizing activity.

In some embodiments the host cell are utilized with an adjuvant.Specific non-limiting examples of adjuvants of use are GM-CSF,Bacillus-Calmette-Guerin adjuvant or CD40 ligand (CD40L).

Attenuated Bacteria: Listeria and Salmonella

Prokaryotic host cells include Listeria and Salmonella, which can beused directly to provide a Brachyury protein and/or Brachyurypolypeptide, such as for use in the methods disclosed herein. Thus, insome embodiments, an attenuated invasive intracellular bacterium capableof infecting a mammalian host or cell thereof, but having a decreasedability in intra- and intercellular movement in the host as compared toa wild type bacterium is utilized. In some embodiments, the bactium istransformed with (a) a promoter activated when said bacterium is presentin the cytosol of a host cell, operably linked to a structural gene orfragment thereof, encoding a polypeptide which is lethal to thebacterium, (b) a host cell-compatible promoter, operably linked to astructural gene or fragment thereof, encoding a Brachyury protein,wherein (a) and (b) can be on the same plasmid or different plasmids or(a) can be integrated into the bacterial chromosome. The attenuatedbacteria include a number of intracellular bacteria, such as Salmonella,Yersinia, Renibacterium and Listeria capable of intracellular growth(Coynault et al., Molecular Microbiology 22, 149-160, 1996; Hohmann etal., Vaccine 14, 19-14, 1996; Karem et al., Infection and Immunity 63,4557-4563, 1995; O'Callaghan et al., Infection and Immunity 56, 419-423,1988; Sigwart et al., Infection and Immunity 57, 1858-61, 1989; andSinha et al., Infection and Immunity 65, 1566-1569, 1997).

The disclosed attenuated invasive bacteria can still invade a host, orcells thereof, but are not pathogenic. In some embodiments, attenuationcan be achieved by mutation (see T. Maniatis, et al. Molecular Cloning:A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., 1989). Inspecific non-limiting examples, attenuation can be caused by mutatingthe bacterial genes which encode for pathogenic and/or toxicpolypeptides. Such a mutation can be achieved randomly, such as bychemical modification and selected later, for example, for loss offunction, or it can be site directed. Thus in some embodiments, thebacterium is attenuated by deletion, insertion or point mutations, toeliminate the function of certain genes that encode polypeptides whichlead to pathogenesis. In some embodiments, the bacterium is attenuatedby deletion of an entire operon by chromosomal deletion, such as theattenuated L. monocytogenes mutant strain DELTA2 that is a derivative ofthe fully virulent wild-type strain EGD, and lacks the entirelecithinase operon consisting of the genes mpl, actA and plcB due to achromosomal deletion. Due to the deletion of this operon theinflammatory response caused by L. monocytogenes during infection of amammalian host is significantly reduced. Suitable bacteria are disclosedfor example, in U.S. Pat. No. 6,143,551, which is incorporated byreference herein.

A variety of potential live Salmonella strains with differentattenuation levels, which subsequently serve as platforms for thedevelopment of recombinant live Salmonella carrier strains that expressheterologous antigens. Such recombinant live Salmonella vaccine carriersare equipped with modules comprising variable gene cassettes thatregulate the expression of heterologous antigens in Salmonella anddetermine presentation of the heterologous antigens to the host immunesystem. By combinations of both systems, differently attenuated liveSalmonella vaccine strains and variable gene cassettes, a variety ofrecombinant live carrier strains can be generated that have broadapplication.

S. typhimurium contains two type III secretion systems for virulencedeterminants. The first controls bacterial invasion of epithelial cells,and is encoded by genes within a 40 kb pathogenicity island (SPI1). Theother is encoded by genes within a second 40 kb pathogenicity island(SPI2) and is required for systemic growth of this pathogen within itshost. The genes located on pathogenicity island SPI1 are mainlyresponsible for early steps of the infection process, the invasion ofnon-phagocytic host cells by the bacterium. For most of the SPI1 genes,mutations result in a reduced invasiveness in vitro. However, mutantsthat are defective in invasion are not necessarily avirulent. Incomparison, virulence studies of SPI2 mutants have shown them to beattenuated by at least five orders of magnitude compared with thewild-type strain after both oral and intraperitoneal inoculation ofmice.

Many of the genes encoding components of the SPI2 secretion system arelocated in a 25 kb segment of SPI2. SPI2 contains genes for a type IIIsecretion apparatus (ssa) and a two component regulatory system (ssr),as well as candidate genes for a set of secreted effectors (sse) andtheir specific chaperones (ssc). On the basis of similarities with genespresent in other bacterial pathogens, the first 13 genes within thessaK/U operon and ssaJ encode components of the secretion systemapparatus. A number of additional genes, including ssaC, which encode asecretion system apparatus protein and a two component regulatoryprotein, respectively, are found in a region approximately 8 kb fromssaJ.

Thus, an attenuated gram-negative cell can have inactivated at least onegene selected from effector (sse) gene secretion apparatus (ssa) genes,chaperon (ssc) genes and regulation (ssr) genes. With regard to the ssegenes are affected by the inactivation, the inactivated gene ispreferably sseC, sseD, sseE or a combination thereof. As far as the ssrgenes are affected by the inactivation, preferably at least ssrB isinactivated. As far as the ssc genes are affected by the inactivation,preferably at least sscB is inactivated.

Attenuation can be the result of original mutations in SPI2 gene locus.Combination of the individual mutations in the SPI2 gene locus with eachother, and with other known attenuating gene mutations, such as aroA,results in a broad repertoire of attenuation and immunogenicity.Different expression cassettes can be introduced on these platforms,allowing further modulation of the immune response directed against theheterologous antigens.

Pathogenic Salmonella or Listeria serve as a basis for the constructionof a panel of different live Salmonella vaccine prototypes generated bygradual attenuations accomplished through the introduction of definedSPI2 gene locus mutations. Each resulting individual live Salmonellavaccine prototype is further transformed into a multivalent recombinantvaccine by the introduction of exchangeable DNA modules carrying (1) anucleic acid encoding a Brachyury protein or polypeptide and (2)adequate expression systems executing efficacious antigen presentationto the host immune system. In concert, these features elicit a specificimmune response, such as a Brachyury specific T cell response.

The inactivation of the gene of the SPI2 locus (or functional homologuethereof in cells other than Salmonella) is effected by a mutation whichmay comprise deletion. In some embodiments, the deletion is cause byinsertion of a heterologous nucleic acid, such as a nucleic acidencoding Brachyury into the gene to be inactivated. With regard toSalmonella, pathogenic Salmonella species are gradually attenuated bymutations in individual virulence genes that are part of the SPI2 genelocus, for example an sse gene coding for an effector protein, such assseC, ssed or sseE, or an ssc gene, such as sscB, coding for achaperone, or an ssr gene, such as ssrB, coding for a regulator.Individual mutation of each of these genes leads to a unique individualgrade of attenuation, which, in turn, effects a characteristic immuneresponse at the mucosal, humoral and cellular levels. The individualgrade of attenuation can be moderately increased by combinations of atleast two gene mutations within the SPI2 gene locus or by combinationwith a mutation in another Salmonella gene known to attenuate virulence,such as an aro gene, for example aroA. A stronger grade of attenuationis achieved by mutation of a virulence gene that is part of apolycistronic gene cluster encoding several virulence factors, such asthe transcriptional unit comprising the sseC, sseD, sseE and sscB genes,such that the mutation exerts a polar effect, disrupting expression ofthe following genes. The grade of attenuation may directly depend on thenumber of virulence genes that are affected by the polar mutation aswell as their individual characteristics. Finally, the strongestattenuation is achieved when regulatory genes, such as ssrB, aremutated. Again, each mode of attenuation of Salmonella leads to thegeneration of a live Salmonella strain that evokes an immune response,see U.S. Pat. No. 7,700,104, which is incorporated herein by reference.

With regard to Listeria, L. monocytogenes is a Gram-positive,facultative intracellular bacterium that lacks lipopolysaccharide (LPS)and is also able to invade a wider range of mammalian cells where itreplicates in the cytosol as well (Portnoy et al., Infect. Immun. 60,1263, 1992). Since it invades its host through the intestinal mucosalsurface, L. monocytogenes is also a candidate for oral vaccination.Shortly after infection, bacteria are found in the spleen whereprofessional APC are abundant. Delivery of DNA to those cells istherefore significantly enhanced by the use of suitably constructed L.monocytogenes. Attenuated L. monocytogenes cells are lysed in thecytosol of the host cell by the production of a P_(actA)-dependent phagelysin releasing plasmid DNA which carries a heterologous gene, such as anucleic acid encoding Brachyury protein, under the control of apromoter, such as, but not limited to, the human cytomegalovirus majorimmediate-early promoter/enhancer region. Beside the advantages ofavoiding the use of antibiotics, lysin-mediated plasmid release is anefficient method comparable to eliminating the bacteria by antibiotictreatment.

The attenuated bacterium can be a mutant of wild-type Listeria whichinvades host cells and is released into the cytosol of the infectedcells with similar efficiencies as the wild-type strain, but is impairedin intra- and intercellular movement. For example, the mutant L.monocytogenes strain DELTA2 is unable to polymerise host cell actin inthe cytosol which L. monocytogenes wild type strain uses for itsmovement inside the host cell. Furthermore, due to the deletion of plcB,the bacterium is unable to lyse the host cell membranes which the wildtype strain lyses upon entering neighboring cells. Mutant bacteria aretherefore unable to move from one infected cell into a neighboring cell(cell-to-cell spread). This illustrates a decreased ability as comparedto wild type strains in intra- and inter-cellular movement. In someembodiments the attenuated bacterium is a mutant of L. monocytogeneswhich invades the host and is released into the cytosol of the infectedcells with similar efficiencies as the wild-type strain, but it is notpathogenic, i.e., it doesn't cause a disease. In specific non-limitingexamples, the bacterium is L. monocytogenes that lacks the entirelecithinase operon containing the genes mpl, actA and plcB, and encodesa Brachyury protein or a Brachyury polypeptide.

In some embodiments, a structural gene or fragment thereof is alsoincluded, such as encoding a polypeptide which is lethal to thebacterium is any polypeptide which when expressed in the bacterium willresult in the release of plasmid DNA and death of the bacterium, forexample by lysis of the bacterium. In some embodiments, the polypeptidecan be a bacteriophage lysin, preferable the gene product of ply 118 orother Listeria-phage-encoded lysins, for example the mureinhydrolaseencoded by the iap gene of L. monocytogenes or other iap-related genesespecially iap of L. grayi. The lysis protein PLY 118 is a late geneproduct of the Listeria bacteriophage A118 necessary for the release ofprogeny phages. PLY 118 is a highly active, cell wall-hydrolyzing enzymespecific for Listeria (Loessner et al., Mol. Microbiol. 16, 1231, 1995).By a promoter activated when it is present in an invasive bacteriumwhich is in the cytosol of a host cell, it is meant any promoter which,when the bacteria is inside the infected cell, is (under the control ofa transcription activator which is) preferentially turned on, drivingits transcription. For example, the L. monocytogenes promoter P_(actA)can be used. The P_(actA) promoter is controlled by the transcriptionactivator PrfA which regulates most of the known virulence genes of L.monocytogenes and is specifically activated in the cytosol of theinfected host cells to interact with the actA promoter. Other promoterswhich can be used are other promoters of L. monocytogenes, such as thosecontrolling the expression of inlC and other genes for small internalins(Engelbrecht et al., Mol. Microbiol. 21:823-837, 1996).

Therapeutic Methods and Pharmaceutical Compositions

The Brachyury proteins and Brachyury polypeptides disclosed herein,nucleic acids encoding the Brachyury proteins or Brachyury polypeptides,or host cells including these nucleic acids can be used to generate animmune response in a subject, such as, but not limited to, a Brachyuryspecific CD4+ T cell response and/or a Brachyury CD8+ T cells response.In several examples, the subject has a cancer that expresses Brachyury.In other embodiments, the method is a method for preventing cancer inthe subject. The subject can be at risk of developing cancer. Inspecific non-limiting examples, the subject has high grade prostaticintraepithelial neoplasia, familial adenomatous polyposis, or atypia ofthe breast. The methods include administering to a subject atherapeutically effective amount of one or more of the Brachyuryproteins and/or polypeptides disclosed herein, nucleic acids encodingthese Brachyury proteins or Brachyury polypeptides, host cells, such asListeria or Salmonella host cells, dendritic cells presenting epitopesof the protein or polypeptide, and/or vectors including these nucleicacids, in order to generate an immune response.

The methods can include selecting a subject in need of treatment, suchas a subject with a cancer that expresses Brachyury or a cancer with thepotential to express Brachyury. In several examples, the methods includeselecting a subject with a cancer of the small intestine, stomach,kidney, bladder, uterus, ovaries, testes lung, colon, prostate, tumor ofB cell origin (such as chronic lymphocytic leukemia (CLL), a B celllymphoma, Burkitt's lymphoma or a Hodgkin's lymphoma) or breast cancerwherein the cancer expresses Brachyury or has the potential to expressBrachyury. In some non-limiting examples, examples, the cancer isradiation resistant and/or chemotherapy resistant. In additionalnon-limiting examples, the subject has breast cancer, such as a ductalcarcinoma, for example an infiltrating ductal carcinoma or an estrogenreceptor negative and progesterone receptor negative breast cancer. Infurther examples, the subject has high-grade prostatic intraepithelialneoplasia, familial adenomatous polyposis, or atypia of the breast.

In exemplary applications, compositions are administered to a subject inan amount sufficient to raise an immune response to Brachyury-expressingcells, such as a CD4+ T cell response. A Brachyury specific CD8+ T cellresponse can also be induced using the methods disclosed herein.Administration induces a sufficient immune response to slow theproliferation of Brachyury-expressing cells, or to inhibit their growth,or to reduce a sign or a symptom of the cancer, or to prevent a cancer.Amounts effective for this use will depend upon the severity of thedisease, the general state of the patient's health, and the robustnessof the patient's immune system. In one example, a therapeuticallyeffective amount of the composition is that which provides eithersubjective relief of a symptom(s) or an objectively identifiableimprovement as noted by the clinician or other qualified observer. Thecomposition can include a Brachyury protein and/or a Brachyurypolypeptide, a nucleic acid encoding a Brachyury protein and/or aBrachyury polypeptide, a vector including the nucleic acid, or a hostcell expressing the Brachyury protein and/or Brachyury polypeptide. Itshould be noted that these compositions can be used in combination.

The composition can be administered by any means known to one of skillin the art (see Banga, A., “Parenteral Controlled Delivery ofTherapeutic Peptides and Proteins,” in Therapeutic Peptides andProteins, Technomic Publishing Co., Inc., Lancaster, Pa., 1995). Thus,the composition can be administered either locally or systemically, suchas by intramuscular, subcutaneous, intraperitoneal or intravenousinjection, but even oral, nasal, transdermal or anal administration iscontemplated. In one embodiment, administration is by subcutaneous orintramuscular injection.

Use of Proteins, Polypeptides, Nucleic Acids and Host Cells

When the Brachyury protein and/or the Brachyury polypeptide isadministered, to extend the time during which protein is available tostimulate a response, the protein and/or polypeptide can be provided asan implant, an oily injection, in a liposome, or as a particulatesystem. The particulate system can be a microparticle, a microcapsule, amicrosphere, a nanocapsule, or similar particle. (see, e.g., Banga,supra). A particulate carrier based on a synthetic polymer has beenshown to act as an adjuvant to enhance the immune response, in additionto providing a controlled release. Adjuvants can also be used incombination with the protein, including, for example, chitosan, aluminumsalts, an immunostimulatory oligodeoxynucletoide, liposomes and/or oneor more cytokines. The Brachyury protein or polypeptide can beadministered in a liposome.

In one specific, non-limiting example, the Brachyury protein isadministered in a manner to direct the immune response to a cellularresponse (that is, a Brachyury specific CD4+ response and/or CD8+response), rather than a humoral (antibody) response. The Brachyurypolypeptide can induce both a Brachyury specific CD4+ T cell responseand a Brachyury specific CD8+ T cell response. Methods for measuring aCD4+ and CD8+ T cell response are known in the art, and includebiological assays, ELISPOT assays, and fluorescence activated cellsorting. An exemplary assay for measuring Brachyury specific CD4+ Tcells is disclosed in the examples below.

In one specific, non-limiting example, a pharmaceutical composition forintravenous administration would include about 0.1 μg to 10 mg ofimmunogenic Brachyury protein and/or Brachyury polypeptide per patientper day. Dosages from 0.1 up to about 100 mg per patient per day can beused, particularly if the agent is administered to a secluded site andnot into the circulatory or lymph system, such as into a body cavity orinto a lumen of an organ. Actual methods for preparing administrablecompositions will be known or apparent to those skilled in the art andare described in more detail in such publications as RemingtonsPharmaceutical Sciences, 19^(th) Ed., Mack Publishing Company, Easton,Pa., 1995.

Optionally, one or more immunostimulatory molecules, such as IL-2, IL-6,IL-12, LFA (for example, LFA-1, LFA-2 and/or LFA-3), CD72, RANTES,G-CSF, GM-CSF, TNF-α, IFN-γ, ICAM-1, B7-1, B7-2, other B7 relatedmolecules, OX-40L and/or or 41 BBL, or combinations of these molecules,can be used as biological adjuvants (see, for example, Salgaller et al.,1998, J. Surg. Oncol. 68(2):122-38; Lotze et al., 2000, Cancer J Sci.Am. 6(Suppl 1):S61-6; Cao et al., 1998, Stem Cells 16(Suppl 1):251-60;Kuiper et al., 2000, Adv. Exp. Med. Biol. 465:381-90). These moleculescan be administered systemically (or locally) to the host. In severalexamples, IL-2, RANTES, GM-CSF, TNF-α, IFN-γ, G-CSF, LFA-3, CD72, B7-1,B7-2, B7-1, B7-2, OX-40L, 41 BBL and/or ICAM-1 are administered. IL-15or an IL-15/IL-15 receptor complex can be administered.

A number of means for inducing cellular responses, both in vitro and invivo, are known. Lipids have been identified as agents capable ofassisting in priming T cells in vivo against various antigens. Forexample, as described in U.S. Pat. No. 5,662,907, palmitic acid residuescan be attached to the alpha and epsilon amino groups of a lysineresidue and then linked (for example, via one or more linking residues,such as glycine, glycine-glycine, serine, serine-serine, or the like) toan immunogenic peptide or protein. The lipidated peptide can then beinjected directly in a micellar form, incorporated in a liposome, oremulsified in an adjuvant. As another example, E. coli lipoproteins,such as tripalmitoyl-S-glycerylcysteinlyseryl-serine can be used toprime tumor specific T cells when covalently attached to an appropriatepeptide or protein (see, Deres et al., Nature 342:561, 1989). Further,as the induction of neutralizing antibodies can also be primed with thesame molecule conjugated to a protein which displays an appropriateepitope, two compositions can be combined to elicit both humoral andcell-mediated responses where that is deemed desirable.

A pharmaceutical composition including a Brachyury protein and/orBrachyury polypeptide is thus provided. These compositions are used togenerate an immune response, such as for immunotherapy.

In one embodiment, the Brachyury protein and/or the Brachyurypolypeptide is mixed with an adjuvant containing two or more of astabilizing detergent, a micelle-forming agent, and an oil. Suitablestabilizing detergents, micelle-forming agents, and oils are detailed inU.S. Pat. Nos. 5,585,103; 5,709,860; 5,270,202; and 5,695,770, all ofwhich are incorporated by reference. A stabilizing detergent is anydetergent that allows the components of the emulsion to remain as astable emulsion. Such detergents include polysorbate, 80 (TWEEN)(Sorbitan-mono-9-octadecenoate-poly(oxy-1,2-ethanediyl; manufactured byICI Americas, Wilmington, Del.), TWEEN 40™, TWEEN 20™, TWEEN 60™,Zwittergent™ 3-12, TEEPOL HB7™, and SPAN 85™. These detergents areusually provided in an amount of approximately 0.05 to 0.5%, such as atabout 0.2%. A micelle forming agent is an agent which is able tostabilize the emulsion formed with the other components such that amicelle-like structure is formed. Such agents generally cause someirritation at the site of injection in order to recruit macrophages toenhance the cellular response. Examples of such agents include polymersurfactants described by BASF Wyandotte publications, e.g., Schmolka, J.Am. Oil. Chem. Soc. 54:110, 1977, and Hunter et al., J. Immunol129:1244, 1981, PLURONIC™ L62LF, L101, and L64, PEG1000, and TETRONIC™1501, 150R1, 701, 901, 1301, and 130R1. The chemical structures of suchagents are well known in the art. In one embodiment, the agent is chosento have a hydrophile-lipophile balance (HLB) of between 0 and 2, asdefined by Hunter and Bennett, J. Immun. 133:3167, 1984. The agent canbe provided in an effective amount, for example between 0.5 and 10%, orin an amount between 1.25 and 5%.

The oil included in the composition is chosen to promote the retentionof the antigen in oil-in-water emulsion, such as to provide a vehiclefor the desired antigen, and preferably has a melting temperature ofless than 65° C. such that emulsion is formed either at room temperature(about 20° C. to 25° C.), or once the temperature of the emulsion isbrought down to room temperature. Examples of such oils includesqualene, Squalane, EICOSANE™, tetratetracontane, glycerol, and peanutoil or other vegetable oils. In one specific, non-limiting example, theoil is provided in an amount between 1 and 10%, or between 2.5 and 5%.The oil should be both biodegradable and biocompatible so that the bodycan break down the oil over time, and so that no adverse affects, suchas granulomas, are evident upon use of the oil.

In one embodiment, the adjuvant is a mixture of stabilizing detergents,micelle-forming agent, and oil available under the name PROVAX® (IDECPharmaceuticals, San Diego, Calif.). In other embodiments, the Brachyuryprotein and/or Brachyury polypeptide are included in a liposome.

Adjuvants can also be administered with the Brachyury protein and/orBrachyury polypeptide. An adjuvant can be any immunostimulatorymolecule, such as a cytokine, immunostimulatory nucleic acid, or abiological adjuvant (see above). The adjuvant can be chitosan. Chitosanis a linear polysaccharide formed from repeating beta (1-4 linked)N-acetyl-D-glucosamine and D-glucosamine units, and is derived from thepartial deacetylation of chitin obtained from the shells of crustaceans.Chitosan can be made commercially by a heterogeneous alkaline hydrolysisof chitin to give a product which possesses a random distribution ofremaining acetyl moieties. The properties of chitosans depend upon interalia the degree of deacetylation, and the molecular weight. Mostcommercially available chitosans contain a population of chitosanmolecules of varying molecular weights and varying concentrations of thecomponent N-acetyl-D-glucosamine and D-glucosamine groups. Theimmunological properties of chitosans are known to be linked to theratio between the N-acetyl-D-glucosamine and D-glucosamine groups. Theefficacy of chitosans as adjuvants depends to a considerable extent onthe extent of the level of deacetylation. Thus, in some embodiments, thechitosan is at least 80% deacetylated, see U.S. Pat. No. 6,534,065,which is incorporated herein by reference.

Controlled release parenteral formulations can be made as implants, oilyinjections, or as particulate systems. For a broad overview of proteindelivery systems, see Banga, Therapeutic Peptides and Proteins:Formulation, Processing, and Delivery Systems, Technomic PublishingCompany, Inc., Lancaster, Pa., 1995. Particulate systems includemicrospheres, microparticles, microcapsules, nanocapsules, nanospheres,and nanoparticles. Microcapsules contain the therapeutic protein as acentral core. In microspheres, the therapeutic agent is dispersedthroughout the particle. Particles, microspheres, and microcapsulessmaller than about 1 μm are generally referred to as nanoparticles,nanospheres, and nanocapsules, respectively. Capillaries have a diameterof approximately 5 μm so that only nanoparticles are administeredintravenously. Microparticles are typically around 100 μm in diameterand are administered subcutaneously or intramuscularly (see Kreuter,Colloidal Drug Delivery Systems, J. Kreuter, ed., Marcel Dekker, Inc.,New York, N.Y., pp. 219-342, 1994; Tice & Tabibi, Treatise on ControlledDrug Delivery, A. Kydonieus, ed., Marcel Dekker, Inc. New York, N.Y.,pp. 315-339, 1992).

Polymers can be used for ion-controlled release. Various degradable andnondegradable polymeric matrices for use in controlled drug delivery areknown in the art (Langer, Accounts Chem. Res. 26:537, 1993). Forexample, the block copolymer, polaxamer 407 exists as a viscous yetmobile liquid at low temperatures but forms a semisolid gel at bodytemperature. It has shown to be an effective vehicle for formulation andsustained delivery of recombinant interleukin-2 and urease (Johnston etal., Pharm. Res. 9:425, 1992; and Pec. J. Parent. Sci. Tech. 44(2):58,1990). Alternatively, hydroxyapatite has been used as a microcarrier forcontrolled release of proteins (Ijntema et al., Int. J. Pharm. 112:215,1994). In yet another aspect, liposomes are used for controlled releaseas well as drug targeting of the lipid-capsulated drug (Betageri et al.,Liposome Drug Delivery Systems, Technomic Publishing Co., Inc.,Lancaster, Pa., 1993). Numerous additional systems for controlleddelivery of therapeutic proteins are known (e.g., U.S. Pat. Nos.5,055,303; 5,188,837; 4,235,871; 4,501,728; 4,837,028; 4,957,735; and5,019,369; 5,055,303; 5,514,670; 5,413,797; 5,268,164; 5,004,697;4,902,505; 5,506,206; 5,271,961; 5,254,342; and 5,534,496).

In another embodiment, the composition includes a nucleic acid encodinga Brachyury protein and/or a Brachyury polypeptide. A therapeuticallyeffective amount of the polynucleotide encoding the Brachyury proteinand/or the Brachyury polypeptide can be administered to a subject inorder to generate an immune response. In one specific, non-limitingexample, a therapeutically effective amount of the polynucleotideencoding the Brachyury protein and/or Brachyury polypeptide isadministered to a subject to treat or prevent cancer.

Optionally, one or more immunostimulatory molecules and/or costimulatorymolecules, such as IL-2, IL-6, IL-12, LFA (for example, LFA-1, LFA-2and/or LFA-3), CD72, RANTES, G-CSF, GM-CSF, TNF-α, IFN-γ ICAM-1, B7-1,B7-2, other B7 related molecules, OX-40L or 41 BBL, or combinations ofthese molecules, can be used as biological adjuvants (see, for example,Salgaller et al., 1998, J. Surg. Oncol. 68(2):122-38; Lotze et al.,2000. Cancer J Sci. Am. 6(Suppl 1):S61-6; Cao et al., 1998, Stem Cells16(Suppl 1):251-60; Kuiper et al., 2000, Adv. Exp. Med. Biol.465:381-90). These molecules can be administered systemically (orlocally) to the host. In several examples, IL-2, RANTES, GM-CSF, TNF-α,IFN-γ, G-CSF, LFA-3, CD72, B7-1, B7-2, B7-1, B7-2, OX-40L, 41 BBL and/orICAM-1 are administered. L-15 or an IL-15/IL-15 receptor complex can beadministered.

Optionally, a non-pox non-yeast vector is administered that encodes oneor more immunostimulatory or costimulatory molecules, such as IL-2,IL-6, IL-12, IL-15, LFA (for example, LFA-1, LFA-2 and/or LFA-3), CD72,RANTES, G-CSF, GM-CSF, TNF-α, IFN-γ, ICAM-1, B7-1, B7-2, other B7related molecules, OX-40L or 41 BBL, or combinations of these molecules(see, for example, Salgaller et al., 1998, J. Surg. Oncol. 68(2):122-38;Lotze et al., 2000, Cancer J Sci. Am. 6(Suppl 1):S61-6; Cao et al.,1998, Stem Cells 16(Suppl 1):251-60; Kuiper et al., 2000, Adv. Exp. Med.Biol. 465:381-90). In several examples, the vector can encode IL-2,RANTES, GM-CSF, TNF-α, IFN-γ, G-CSF, LFA-3, CD72, B7-1, B7-2, B7-1,B7-2, OX-40L, 41 BBL and/or ICAM-1. In various embodiments, the nucleicacid encoding the biological adjuvant can be cloned into same vector asthe Brachyury protein or Brachyury polypeptide coding sequence, or thenucleic acid can be cloned into one or more separate vectors forco-administration. In addition, nonspecific immunomodulating factorssuch as Bacillus Cahnette-Guerin (BCG) and levamisole can beco-administered.

One approach to administration of nucleic acids is direct immunizationwith plasmid DNA, such as with a mammalian expression plasmid. Asdescribed above, the nucleotide sequence encoding a Brachyury protein orpolypeptide can be placed under the control of a promoter to increaseexpression of the molecule.

Immunization by nucleic acid constructs is well known in the art andtaught, for example, in U.S. Pat. No. 5,643,578 (which describes methodsof immunizing vertebrates by introducing DNA encoding a desired antigento elicit a cell-mediated or a humoral response), and U.S. Pat. Nos.5,593,972 and 5,817,637 (which describe operably linking a nucleic acidsequence encoding an antigen to regulatory sequences enablingexpression). U.S. Pat. No. 5,880,103 describes several methods ofdelivery of nucleic acids encoding immunogenic peptides or otherantigens to an organism. The methods include liposomal delivery of thenucleic acids (or of the synthetic peptides themselves), andimmune-stimulating constructs, or ISCOMS™, negatively charged cage-likestructures of 30-40 nm in size formed spontaneously on mixingcholesterol and Quil A™ (saponin). Protective immunity has beengenerated in a variety of experimental models of infection, includingtoxoplasmosis and Epstein-Barr virus-induced tumors, using ISCOMS™ asthe delivery vehicle for antigens (Mowat and Donachie, Immunol. Today12:383, 1991). Doses of antigen as low as 1 μg encapsulated in ISCOMS™have been found to produce Class I mediated CTL responses (Takahashi etal., Nature 344:873, 1990). In other embodiments, the nucleic acid canbe loaded onto gold microspheres by standard methods and introduced intothe skin by a device such as Bio-Rad's HELIOS™ Gene Gun. The nucleicacids can be “naked,” consisting of plasmids under control of a strongpromoter. Typically, the DNA is injected into muscle, although it canalso be injected directly into other sites, including tissues inproximity to metastases. Dosages for injection are usually around 0.5μg/kg to about 50 mg/kg, and typically are about 0.005 mg/kg to about 5mg/kg (see, for example, U.S. Pat. No. 5,589,466).

In another approach to using nucleic acids for immunization, a Brachyuryprotein or Brachyury polypeptide can also be expressed by attenuatedhost cells or non-pox non-yeast viral vectors. These vectors and hostcells are disclosed above. Suitable non-pox non-yeast viral vector,include an adenovirus, an alphavirus, a lentivirus, a measles virus or apoliovirus vector. Suitable host cell include an attenuated bacterium,such as Listeria or Salmonella host cells.

A first recombinant non-pox virus encoding a Brachyury protein can beused in conjunction with a second recombinant non-pox virus which hasincorporated into a viral genome or infectable portion thereof one ormore genes or DNA sequences encoding B7-1, B7-2, or B7-1 and B7-2,wherein the composition is able to coinfect a host cell resulting incoexpression of the polypeptide and the B7-1, B7-2, or B7-1 and B7-2encoding genes or DNA sequences (see U.S. Pat. Nos. 6,893,869, and6,045,908, which are incorporated by reference herein). The expressionof the B7 gene family has been shown to be an important mechanism ofanti-tumor responses in both mice and humans.

When a non-pox viral vector is utilized, it is desirable to provide therecipient with a dosage of each recombinant virus in the composition inthe range of from about 10⁵ to about 10¹⁰ plaque forming units/mgmammal, although a lower or higher dose can be administered. Thecomposition of recombinant viral vectors can be introduced into a mammaleither prior to any evidence of a cancer, or to mediate regression ofthe disease in a mammal afflicted with the cancer. Examples of methodsfor administering the composition into mammals include, but are notlimited to, exposure of cells to the recombinant virus ex vivo, orinjection of the composition into the affected tissue or intravenous,subcutaneous, intradermal or intramuscular administration of the virus.Alternatively the recombinant viral vector or combination of recombinantviral vectors may be administered locally by direct injection into thecancerous lesion in a pharmaceutically acceptable carrier. Generally,the quantity of recombinant non-pox viral vector, carrying the nucleicacid sequence of a Brachyury protein or Brachyury polypeptide to beadministered is based on the titer of virus particles. An exemplaryrange of the immunogen to be administered is 10⁵ to 10¹⁰ virus particlesper mammal, such as a human.

In the embodiment where a combination of a first recombinant viralvector carrying a nucleic acid sequence of a Brachyury protein orBrachyury polypeptide and a second recombinant viral vector carrying thenucleic acid sequence of one or more costimulatory or immunostimulatorymolecules is used, the mammal can be immunized with different ratios ofthe first and second recombinant viral vector. In one embodiment theratio of the first vector to the second vector is about 1:1, or about1:3, or about 1:5. Optimal ratios of the first vector to the secondvector may easily be titered using the methods known in the art (see,for example, U.S. Pat. No. 6,893,869, incorporated herein by reference).Simultaneous production of an immunostimulatory molecule and theBrachyury protein or Brachyury polypeptide enhances the generation ofspecific effectors. Without being bound by theory, dependent upon thespecific immunostimulatory molecules, different mechanisms might beresponsible for the enhanced immunogenicity: augmentation of help signal(IL-2), recruitment of professional APC (GM-CSF), increase in CTLfrequency (IL-2), effect on antigen processing pathway and MHCexpression (IFNγ and TNFα) and the like. For example, IL-2, IL-6, IL-15,interferon, tumor necrosis factor, or a nucleic acid encoding thesemolecules, can be administered in conjunction with a Brachyury protein,or a nucleic acid encoding a Brachyury protein or Brachyury polypeptide.The co-expression of a Brachyury protein or Brachyury polypeptidetogether with at least one immunostimulatory molecule can be effectivein an animal model to show anti-tumor effects.

Single or multiple administrations of the compositions are administereddepending on the dosage and frequency as required and tolerated by thesubject. In one embodiment, the dosage is administered once as a bolus,but in another embodiment can be applied periodically until atherapeutic result is achieved. Generally, the dose is sufficient toinduce an immune response to Brachyury, to treat or ameliorate symptomsor signs of disease, without producing unacceptable toxicity to thesubject. Systemic or local administration can be utilized.

Use of Antigen Presenting Cells

In another method, antigen presenting cells (APCs), such as dendriticcells, are pulsed or co-incubated with a Brachyury protein and/orBrachyury polypeptide in vitro. In one specific, non-limiting example,the antigen presenting cells can be autologous cells. A therapeuticallyeffective amount of the antigen presenting cells, such as dendriticcells presenting epitopes of the protein or polypeptide can then beadministered to a subject. In some embodiments, the method is a methodof treating and/or preventing cancer in a subject.

The Brachyury protein and/or Brachyury polypeptide can be delivered tothe dendritic cells or to dendritic cell precursors via any method knownin the art, including, but not limited to, pulsing dendritic cellsdirectly with antigen, or utilizing a broad variety of antigen deliveryvehicles, such as, for example, liposomes, or other vectors known todeliver antigen to cells. In one specific, non-limiting example anantigenic formulation includes about 0.1 μg to about 1,000 μg, or about1 to about 100 μg of a selected Brachyury protein. The Brachyury proteincan also be administered with agents that promote dendritic cellmaturation. Specific, non-limiting examples of agents of use areinterleukin-4 (IL-4) and granulocyte/macrophage colony stimulatingfactor (GM-CSF), or flt-3 ligand (flt-3L). The preparation can alsocontain buffers, excipients, and preservatives, amongst otheringredients.

In one embodiment, mature antigen presenting cells are generated topresent the Brachyury protein and/or Brachyury polypeptide epitopes.These dendritic cells are then administered alone (or in combinationwith another agent) to a subject with a cancer that expresses Brachyury,or has the potential to express Brachyury, such as a small intestine,stomach, kidney, bladder, uterus, ovary, testis, lung colon, prostatecancer, a tumor of B cell origin (such as chronic lymphocytic leukemia(CLL), a B cell lymphoma, Burkitt's lymphoma or a Hodgkin's lymphoma) orbreast cancer, such as an infiltrating ductal carcinoma or an estrogenreceptor negative and progesterone receptor negative breast cancer. Insome examples, the cancer is radiation resistant and/or chemotherapyresistant. The antigen presenting cells can also be administered toprevent these cancers. The subject can have high grade prostaticintraepithelial neoplasia, familial adenomatous polyposis, or atypia ofthe breast.

The cells can be administered to a subject to inhibit the growth ofcells of Brachyury expressing cancer or a cancer that has the potentialto express Brachyury. In these applications, a therapeutically effectiveamount of activated antigen presenting cells are administered to asubject suffering from a disease, in an amount sufficient to raise animmune response to Brachyury-expressing cells. The resulting immuneresponse is sufficient to slow the proliferation of such cells or toinhibit their growth, or to reduce a sign or a symptom of the tumor.

In a supplemental method, any of these immunotherapies is augmented byadministering a cytokine, such as interleukin (IL)-2, IL-3, IL-6, IL-10,IL-12, IL-15, GM-CSF, or interferons. In another embodiment, the maturedendritic cells are administered in conjunction with a chemotherapeuticagent.

Combination Therapy

In some embodiments, the subject is administered a Brachyury protein, aBrachyury polypeptide, a nucleic acid encoding a Brachyury protein, ahost cell expressing the Brachyury protein, and/or dendritic cells, andis administered an additional agent. In one example, this administrationis sequential. However, the administration can be simultaneous.

In some embodiments the subject has cancer. The cancer can expressBrachyury or have the potential to express Brachyury. Thus, theadditional agent can be a chemotherapeutic agent. Additional agentsinclude radiation, small molecule targeted therapies, monoclonalantibodies, and/or checkpoint inhibitors, such as anti-PD-1, anti-PD-L1,anti-CTLA-4, and others. In some embodiments, the subject isadministered an epithelial growth factor receptor inhibitor, atransforming growth factor (TGF)-β inhibitor, or a tyrosine kinaseinhibitor.

In some embodiments, the additional chemotherapeutic agent is anepithelial growth factor receptor (EGFR) inhibitor. Numerous compoundsare known that inhibit EGFR, see for example, U.S. Pat. Nos. 5,196,446;5,217,999; 5,459,061; 7,049,410; 6,355,678, which are incorporatedherein by reference. A number of EGFR inhibitors are in clinicaldevelopment, such as for the treatment of lung cancer. These includetustuzumab, lapatinib, pertuzumab, panitumumab, genfitinib (IRESSA®),erlotinib (TARCEVA®), cetuximab (ERTIBUX®), afatinib, necitumumab,nimotuzumab, PF299804 (Pfizer), RO5083945 (Roche), ABT-806 (Abbott) andAP2113 (Ariad). In other embodiments, the additional chemotherapeuticagent is a tyrosine kinase inhibitor. These include, but are not limitedto avastin and termsirolimus.

Examples of additional agents of use are alkylating agents,antimetabolites, natural products, or hormones and their antagonists.Examples of alkylating agents include nitrogen mustards (such asmechlorethamine, cyclophosphamide, melphalan, uracil mustard orchlorambucil), alkyl sulfonates (such as busulfan), nitrosoureas (suchas carmustine, lomustine, semustine, streptozocin, or dacarbazine).Examples of antimetabolites include folic acid analogs (such asmethotrexate), pyrimidine analogs (such as 5-FU or cytarabine), andpurine analogs, such as mercaptopurine or thioguanine. Examples ofnatural products include vinca alkaloids (such as vinblastine,vincristine, or vindesine), epipodophyllotoxins (such as etoposide orteniposide), antibiotics (such as dactinomycin, daunorubicin,doxorubicin, bleomycin, plicamycin, or mitocycin C), and enzymes (suchas L-asparaginase). Examples of miscellaneous agents include platinumcoordination complexes (such as cis-diamine-dichloroplatinum II alsoknown as cisplatin), substituted ureas (such as hydroxyurea), methylhydrazine derivatives (such as procarbazine), and adrenocorticalsuppressants (such as mitotane and aminoglutethimide). Examples ofhormones and antagonists include adrenocorticosteroids (such asprednisone), progestins (such as hydroxyprogesterone caproate,medroxyprogesterone acetate, and megestrol acetate), estrogens (such asdiethylstilbestrol and ethinyl estradiol), antiestrogens (such astamoxifen), and androgens (such as testosterone proprionate andfluoxymesterone). Examples of the most commonly used chemotherapy drugsthat can be concurrently administered with the disclosed immunotherapyinclude Adriamycin, Alkeran, Ara-C, BiCNU, Busulfan, CCNU,Carboplatinum, Cisplatinum, Cytoxan, Daunorubicin, DTIC, 5-FU,Fludarabine, Hydrea, Idarubicin, Ifosfamide, Methotrexate, Mithramycin,Mitomycin, Mitoxantrone, Nitrogen Mustard, Taxol (or other taxanes, suchas docetaxel), Velban, Vincristine, VP-16, while some more newer drugsinclude Gemcitabine (Gemzar), Herceptin, Irinotecan (Camptosar, CPT-11),Leustatin, Navelbine, Rituxan STI-571, Taxotere. Topotecan (Hycamtin).Xeloda (Capecitabine), Zevelin and calcitriol. Non-limiting examples ofimmunomodulators that can be used include AS-101 (Wyeth-Ayerst Labs.),bropirimine (Upjohn), gamma interferon (Genentech), GM-CSF (granulocytemacrophage colony stimulating factor, Genetics Institute), IL-2 (Cetusor Hoffman-LaRoche), human immune globulin (Cutter Biological), IMREG(from Imreg of New Orleans, La.), SK&F 106528, and TNF (tumor necrosisfactor; Genentech).

The additional agent can be a small molecule, a vaccine, or a biologic.For example, when the additional agent is a vaccine, the vaccine can bea yeast-based or viral-based (e.g., poxviral-based) vaccine. Examples ofviral vectors include poxvirus, retrovirus, adenovirus, adeno-associatedvirus, herpes virus, polio virus, alphavirus, baculorvirus, and Sindbisvirus. In one embodiment, the viral vector is a poxvirus selected fromthe group consisting of orthopox, avipox, fowlpox, raccoon pox, rabbitpox, capripox (e.g., sheep pox), leporipox, and suipox (e.g., swinepox).Examples of avipox viruses include fowlpox, pigeonpox, and canarypox,such as ALVAC. Examples of orthopox viruses include vaccinia, modifiedvaccinia Ankara (MVA), Wyeth, NYVAC, TROYVAC, Dry-Vax, POXVAC-TC(Schering-Plough Corporation), and derivatives thereof. For example,derivatives of the Wyeth strain include, but are not limited to,derivatives which lack a functional K1L gene.

The vaccine can encode any suitable antigen, such as the Brachyuryprotein or Brachyury polypeptide described herein, 5-α-reductase,α-fetoprotein (“AFP”), AM-1, APC, April, B melanoma antigen gene(“BAGE”), β-catenin, Bcl12, bcr-abl, Brachyury, CA-125, caspase-8(“CASP-8”, also known as “FLICE”), Cathepsins, CD19, CD20,CD21/complement receptor 2 (“CR2”), CD22/BL-CAM, CD23/F_(c)εRII, CD33,CD35/complement receptor 1 (“CR1”), CD44/PGP-1, CD45/leucocyte commonantigen (“LCA”), CD46/membrane cofactor protein (“MCP”), CD52/CAMPATH-1,CD55/decay accelerating factor (“DAF”), CD59/protectin, CDC27, CDK4,carcinoembryonic antigen (“CEA”), c-myc, cyclooxygenase-2 (“cox-2”),deleted in colorectal cancer gene (“DCC”), DcR3, E6/E7, CGFR, EMBP,Dna78, farnesyl transferase, fibroblast growth factor-8a (“FGF8a”),fibroblast growth factor-8b (“FGF8b”), FLK-1/KDR, folic acid receptor,G250, G melanoma antigen gene family (“GAGE-family”), gastrin 17,gastrin-releasing hormone, ganglioside 2 (“GD2”)/ganglioside 3(“GD3”)/ganglioside-monosialic acid-2 (“GM2”), gonadotropin releasinghormone (“GnRH”), UDP-GlcNAc:R₁Man(α1-6)R₂ [GlcNAc toMan(α1-6)]β1,6-N-acetylglucosaminyltransferase V (“GnT V”), GP1,gp100/Pme 17, gp-100-in4, gp15, gp75/tyrosine-related protein-1(“gp75/TRP-1”), human chorionic gonadotropin (“hCG”), heparanase,Her2/neu, human mammary tumor virus (“HMTV”), 70 kiloDalton heat-shockprotein (“HSP70”), human telomerase reverse transcriptase (“hTERT”),insulin-like growth factor receptor-1 (“IGFR-1”), interleukin-13receptor (“IL-13R”), inducible nitric oxide synthase (“iNOS”), Ki67,KIAA0205, K-ras, H-ras, N-ras, KSA, LKLR-FUT, melanoma antigen-encodingfamily (“MAGE-family”, including at least MAGE-1, MAGE-2, MAGE-3, andMAGE-4), mammaglobin, MAP17, Melan-A/melanoma antigen recognized byT-cells-1 (“MART-1”), mesothelin, MIC A/B, MT-MMPs, mucin,testes-specific antigen NY-ESO-1, osteonectin, p15, P170/MDR1, p53,p97/melanotransferrin, PAI-1, platelet-derived growth factor (“PDGF”),μPA, PRAME, probasin, progenipoietin, prostate-specific antigen (“PSA”),prostate-specific membrane antigen (“PSMA”), RAGE-1, Rb, RCAS1, SART-1,SSX-family, STAT3, STn, TAG-72, transforming growth factor-alpha(“TGF-α”), transforming growth factor-beta (“TGF-β”), Thymosin-beta-15,tumor necrosis factor-alpha (“TNF-α”), TP1, TRP-2, tyrosinase, vascularendothelial growth factor (“VEGF”), ZAG, p16INK4, and/orglutathione-S-transferase (“GST”).

In one embodiment, the vaccine is PROSTVAC™, which is a sequentiallydosed combination of two different poxviruses each encoding prostatespecific antigen (PSA) plus three immune enhancing co-stimulatorymolecules, B7.1, ICAM-1, and LFA-3 (TRICOM). The first poxvirus isVaccinia-PSA-TRICOM, and the second poxvirus is Fowlpox-PSA-TRICOM.

Thus, the invention provides a method for treating or preventing cancerin a subject comprising administering to the subject a combinationtherapy comprising:

(1) (a) a protein comprising an amino acid sequence at least 90%identical to the amino acid sequence set forth as SEQ ID NO: 1; (b) apolypeptide comprising at least 15 consecutive amino acids of the aminoacid sequence set forth at SEQ ID NO: 1; (c) a nucleic acid encoding theprotein or the polypeptide; (d) a host cell expressing the protein orthe polypeptide; or (e) a non-pox non-yeast vector encoding the proteinor the polypeptide, and

(2) a small molecule, a vaccine (e.g., a pox-viral or yeast vaccine asdescribed above), or a biologic,

thereby treating or preventing cancer in the subject.

EXAMPLES Example 1: Induction of CD4+ Cells Using Brachyury Protein orBrachyury Polypeptide

FIGS. 1 and 2 show the induction of CD4+ cells using Brachyury proteinand a Brachyury polypeptide.

I. FIG. 1

Methods:

Dendritic cells (DCs) from 2 normal donors were prepared from theadherent cell fraction of peripheral blood mononuclear cells (PBMCs) byculture in the presence of GM-CSF and IL-4. On day 5, a purified,recombinant full length Brachyury protein was added to the cultures (10μg/ml) for 48 hours. For donor 2, an additional culture was set up usingpurified HSA (human serum albumin) control protein (10 μg/ml). On day 7,protein-pulsed DCs were harvested, irradiated (20 Gy) and used asantigen-presenting cells (APCs) to stimulate autologous PBMCs (ratioDC:PBMCs equal to 1:10). On days 3 and 5, IL-2 (20 U/ml) was added tothe cultures. Cells were harvested on day 7 and CD4+ T cells wereisolated by negative selection utilizing CD4 purification magnetic beads(Miltenyi Biotec). CD4+ T cells were subsequently stimulated in asimilar manner for an additional 7-day cycle. On day 7, CD4+ T cellswere re-purified by using CD4 purification magnetic beads, and evaluatedfor IFN-gamma production in response to autologous, irradiated PBMCs(ratio PBMCs:CD4+ T cells equal to 3:1) alone or pulsed with control HSAprotein vs. Brachyury protein (10 μg/ml). Culture supernatants werecollected at 96 hours and evaluated for IFN-gamma by ELISA.

Summary of Findings:

As shown in FIG. 1, Brachyury-specific CD4+ T cells can be expanded fromthe blood of normal donors by culture of PBMCs in the presence ofautologous DCs pulsed with a purified, full length Brachyury protein.After 2 rounds of in vitro stimulation, Brachyury-specific CD4+ T cellsspecifically released IFN-gamma in response to stimulation withautologous PBMCs pulsed with purified Brachyury protein (full length,recombinant) but not with a control, irrelevant protein (HAS).

II. FIG. 2

Methods:

A CD4+ Brachyury-specific T-cell line (T-BRA) was generated from aprostate patient vaccinated with a PSA-based vaccine. CD40L-maturedautologous DCs were used as antigen-presenting cells (APCs). PBMCsobtained on day 90 post-vaccination were added to the APCs and pulsedwith 10 μg/mL of Brachyury 9-mer agonist peptide (WLLPGTSTV) at aneffector:APC ratio of 10:1. The culture was then incubated for 3 days at37° C. in a humidified atmosphere containing 5% CO₂. The culture wasthen supplemented with recombinant human IL-7 and IL-15 at aconcentration of 10 ng/ml for 5 days. The 3-day incubation with peptideand 5-day IL-7 and IL-15 supplement constituted one in vitro stimulation(IVS) cycle. Autologous DCs were used as APCs for 3 in vitro stimulation(IVS) cycles. Irradiated (23,000 rads) autologous EBV-transformed Bcells were used as APCs after the third IVS cycle. For re-stimulationwith EBV-transformed B cells, peptides at a concentration of 10 μg/mLwere used to pulse the autologous EBV-transformed B cells at aneffector:APC ratio of 1:3. CD4+ T cells were then isolated from the cellculture at the end of the 4th IVS and stimulated with a Brachyury classII 15-mer peptide (Brachyury class IIB)(QWGWLLPGTSTL). The cultures werethen supplemented with recombinant human IL-7 and IL-15 at aconcentration of 10 ng/mL for 5 days. The CD4+ T cell line was thenassayed for specificity to the Brachyury class IIB epitope bystimulation with APCs pulsed with the Brachyury class IIB or a controlBrachyury class IIA epitope.

Example 2: Brachyury in Carcinoma

FIGS. 3-7 show the results achieved.

TABLE 1 Expression of Brachyury in primary breast carcinoma tissues byimmunohistochemistry utilizing a murine monoclonal anti-brachyury AbTumor Adjacent Distal Pt # % Positivity Intensity tissue tissue 1 80 ++pos neg 2 80 ++ pos neg 3 90 ++ pos neg 4 90 + neg neg 5 90 + pos neg 630 + neg neg 7 15 +++ neg neg 8 60 + neg neg 9 focal + neg neg 10 70 +neg neg 11 40 + neg neg 12 30 + neg neg 13 focal + neg neg 14 90 +++ negneg 15 90 −/+ pos neg 16 30 + neg neg 17 neg neg neg neg 18 50 ++ negneg 19 neg neg neg neg 20 neg neg neg neg 21 40 ++ pos neg 22 30 ++ posneg 23 85 ++ pos neg 24 80 +++ pos neg 25 30 + pos neg 26 50 ++ pos neg27 65 ++ pos NA 28 40 +++ pos neg 29 25 ++ pos NA 30 15 + pos NA Pos =positive; neg = negative; NA = not available

TABLE 2 Brachyury expression in primary breast carcinoma tissues byimmunohistochemistry by lymph node status, tumor grade, and hormonereceptor expression Number of Tumor tissue tumors positive sample forBrachyury Lymph node status: Node-negative 13/15 (86.7%)* Node-positive10/11 (93.3%) Grade: G1 2/3 (66.7%) G2 — G3 25/27 (92.6%) ER/PRexpression: ER+ PR+ 4/6 (66.7%) ER− PR− 21/22 (95.5%) ER/PR/HER2expression: ER+ PR+ HER2+ 1/2 (50.0%) ER+ PR+ HER2− 3/4 (75.0%) ER− PR−HER2+ 12/12 (100.0%) ER− PR− HER2− 9/10 (90.0%) Statistical analysis wasperformed, comparing node-positive vs. node-negative, grade 3 vs. grade1, and ER− PR− vs. ER+ PR+ samples. *Numbers in parentheses indicatepercentage.

TABLE 3 Expression of Brachyury in metastatic breast carcinoma lesionsby immunohistochemistry using a murine monoclonal anti-brachyury AbBrachyury Pt # Site % Positivity Intensity 6 Breast primary tumor 30 + 6Met⁺ lymph node (a) 90 + 6 Met⁺ lymph node (b) 90 + 6 Non-met lymph nodeneg neg 9 Breast primary tumor focal + 9 Met⁺ lymph node (a) 60 ++ 9Met⁺ lymph node (b) 60 ++ 9 Non-met lymph node neg neg 31 Pleura 90 + 32Bone 90 ++ 33 Bone 90 + 34 Brain 70 ++

Human matched breast primary tumor tissues and metastatic lymph nodesfrom two patients were analyzed for Brachyury expression byimmunohistochemistry. The breast primary tumor tissue samples were frominfiltrating ductal adenocarcinomas. Two lymph nodes positive formetastasis from each patient (a, b) and 1 lymph node negative formetastasis from same patient (c) were assayed. Metastatic lesions fromadditional patients were also analyzed for Brachyury expression byimmunohistochemistry. Adjacent and distal breast tissues in the sampleswere negative for Brachyury expression.

The breast primary tumor tissue samples were from 30 infiltrating ductaladenocarcinomas. Brachyury expression for each sample is reported asstaining in tumor cells (% positivity, intensity), staining in breastadjacent tissue, and staining in breast distal tissue.

In view of the many possible embodiments to which the principles of thedisclosed invention may be applied, it should be recognized that theillustrated embodiments are only preferred examples of the invention andshould not be taken as limiting the scope of the invention. Rather, thescope of the invention is defined by the following claims. We thereforeclaim as our invention all that comes within the scope and spirit ofthese claims.

We claim:
 1. A method for inducing a Brachyury specific CD4+ T cellresponse, the method comprising administering to a subject an effectiveamount of an adenoviral vector encoding (i) a polypeptide comprising SEQID NO: 5 or (ii) a polypeptide consisting of SEQ ID NO: 6, and measuringthe Brachyury specific CD4+ T cell response.
 2. The method of claim 1,further comprising measuring a Brachyury specific CD8+ T cell response.3. The method of claim 1, wherein the subject is human.
 4. The method ofclaim 1, wherein the subject has cancer.
 5. The method of claim 4,wherein the cancer is a breast cancer, small intestine cancer, stomachcancer, kidney cancer, bladder cancer, uterus cancer, ovarian cancer,testes cancer, lung cancer, colon cancer, prostate cancer, chroniclymphocytic leukemia (CLL), a B cell lymphoma, a Burkitt's lymphoma or aHodgkin's lymphoma.
 6. The method of claim 1, comprising administeringto the subject an effective amount of the adenoviral vector encoding theprotein-sufficient to induce Brachyury specific CD4+ T cells and/or CD8+T cells.
 7. The method of claim 1, wherein the adenoviral vector encodesa costimulatory molecule.
 8. The method of claim 7, wherein thecostimulatory molecule is one or more of B7-1, B7-2, LFA-3 or ICAM-1. 9.The method of claim 1, wherein the adenoviral vector comprises a DNAsequence encoding an immunostimulatory molecule, wherein theimmunostimulatory molecule is selected from the group consisting ofIL-2, ICAM-1, LFA-3, CD72, GM-CSF, TNF-α, IFN-γ, IL-12, and IL-6. 10.The method of claim 1, further comprising administering to the subjectan effective amount of an adjuvant.
 11. The method of claim 10, whereinthe adjuvant is chitosan.
 12. The method of claim 1, further comprisingadministering to the subject a therapeutically effective amount of anagent selected from the group consisting of a chemotherapeutic agent,radiation, a small molecule targeted therapeutic, and monoclonalantibodies.
 13. The method of claim 12, wherein the agent is anepithelial growth factor receptor inhibitor, a transforming growthfactor (TGF)-β inhibitor, or a tyrosine kinase inhibitor.
 14. The methodof claim 1, wherein the subject has cancer, and wherein the cancer is achemotherapy resistant cancer or a radiation resistant cancer.
 15. Themethod of claim 1, wherein the adenoviral vector encodes the polypeptidecomprising SEQ ID NO:
 5. 16. The method of claim 1, wherein theadenoviral vector encodes the polypeptide consisting of SEQ ID NO: 6.